Bull. Chem. Soc. Ethiop. 2023, 37(1), 11-21.

ISSN 1011-3924
 2023 Chemical Society of Ethiopia and The Authors Printed in Ethiopia
DOI: https://dx.doi.org/10.4314/bcse.v37i1.2 Online ISSN 1726-801X

SPECTROPHOTOMETRIC DETECTION OF URIC ACID WITH ENZYME-LIKE

REACTION MEDIATED 3,3′,5,5′-TETRAMETHYLBENZIDINE OXIDATION

Jin Yang, Shi Qi Cheng, Rui Shi, Shang Ying Qin, Li Huang and Yi Lin Wang*

School of Chemistry and Chemical Engineering, Guangxi Key Laboratory of Electrochemical

Energy Materials, Guangxi University, Nanning 530004, China

(Received April 27, 2022; Revised August 8, 2022; Accepted August 9, 2022)

ABSTRACT. WO3 nanosheets (NSs) were prepared and characterized by X-ray photoelectron spectrometer
(XPS), X-ray diffractometer (XRD), scanning electron microscope (SEM) and transmission electron microscope
(TEM). The obtained WO3 NSs exhibited peroxidase-like catalytic activity, which can catalyze H2O2 to oxidize
3,3 ',5,5 '-tetramethylbenzidine (TMB) to generate oxidized TMB (oxTMB) with an absorption peak centered at 652
nm. Based on this, a facile method for the spectrophotometric determination of H2O2 was established. Under the
selected conditions, the increase in absorbance of oxTMB enabled the detection of H2O2 ranging from 2.0 to 180
μM. Considering the fact that H2O2 is one of the products of urate oxidase (UAO)-catalyzed uric acid (UA)
oxidation, a convenient method for the selective determination of UA was further developed with the help of UV–
Vis spectrophotometer. The increase of absorbance at 652 nm showed a linear response to UA concentration over
the range of 2.0–180 μM. The limit of detection for UA was as low as 1.25 μM. More importantly, the proposed
method was applied to the determination of UA in serum samples with satisfactory results.

KEY WORDS: Spectrophotometric, WO3 nanosheets, Uric acid, Determination

INTRODUCTION

Uric acid (UA), the end product of purine metabolism, is one of the main antioxidants in human
body, which can be detected in serum and urine [1, 2]. When the serum UA content exceeds 0.46
mM [2], it is considered to be high UA, which is identified as to be a risk signal for various
diseases [3] including cardiovascular disease, kidney disease and metabolic syndrome [4-6]. On
the contrary, a low level (below 0.12 mM) of UA in human blood indicates signs of Parkinson's
disease or multiple sclerosis [4, 5]. Therefore, accurate determination of UA in serum is of great
importance for disease diagnosis. So far, different analysis techniques have been used for the
detection of UA in biological samples, including fluorescence [7, 8], high-performance liquid
chromatography (HPLC) [9, 10], electrochemical [11] and so on. It is worth noting that
fluorescence and HPLC require expensive instruments. Electrochemical method faces
complicated electrode modification process. Compared with these methods, colorimetry
possesses the advantages of simplicity, rapidity and practicality. It can be used to determine
analytes with naked eyes or UV-Vis spectrometer [12-15]. Horseradish peroxidase (HRP) was
used in the traditional colorimetry for the determination of H2O2 and its related substances such
as glucose and UA [16, 17]. Although HRP has the advantages of high catalytic activity and strong
specificity, its disadvantages of high cost and poor stability hinder its wide applications [14, 18].
In recent years, seeking substitutes for natural enzymes has attracted extensive attention. In
2007, it was found for the first time that Fe3O4 nanoparticles (NPs) could catalyze H2O2 to oxidize
3,3',5,5'-tetramethylbenzidine (TMB) [19]. That is, Fe3O4 NPs can be used as peroxidase mimetic.
This innovative application inspired the research of using nanomaterials as mimic enzymes. Since
then, lots of nanomaterials have been reported to exhibit peroxidase (or oxidase)-like activity,
including noble metals (e.g. Au@Ag NPs [20], AgNPs@ZnMOF [21], and Ni-Pt NPs [22]),
transition metal oxides or sulfides (e.g. MnO2 NSs [23], CuO NFs [24], CeO2 NPs [25], and MoS2
__________
*Corresponding author. E-mail: [email protected]
This work is licensed under the Creative Commons Attribution 4.0 International License
12 Jin Yang

NSs [26]), carbon-based nanomaterials (e.g. CQDs [27] and GQDs [28]), etc. As a typical
transition metal oxide, WO3 nanomaterials has advantages of high catalytic activity and good
chemical stability. Huang’s group established a colorimetric sensing platform for H2O2, ascorbic
acid and dopamine detection based on WO3 nanowires (NWs) with peroxidase-like activity [29].
As a peroxidase mimetic, WO3 quantum dots (QDs) have been applied to construct cholesterol
colorimetric sensor [30]. Because of its excellent peroxidase activity, WO3 nanosheets (NSs) were
also applied for the colorimetric determination of xanthine [31]. In the present study, WO3 NSs
were prepared from Na2WO4·2H2O and HNO3 at room temperature. Based on the peroxidase-like
activity of the WO3 NSs and urate oxidase (UAO)-catalyzed uric acid (UA) oxidation, a
convenient and selective colorimetric method was developed for the sensitive detection of UA
(Scheme 1).

Scheme 1. Schematic diagram of WO3 NSs preparation and uric acid determination.

EXPERIMENTAL

Reagents and apparatus

Sodium tungstate (Na2WO4·2H2O), hydrogen peroxide (H2O2), 3,3',5,5'-tetramethylbenzidine

(TMB), isopropanol (IPA) and methanol (MA) were provided by Tianjin Da Mao Chemical
Reagent Factory (Tianjin, China). Uric acid (UA), urate oxidase (UAO), glucose (Glu), fructose
(Fru), lysine (Lys), threonine (Thr), lactose (Lac) and glycine (Gly) were purchased from
Shanghai Maclean Biochemical Co., Ltd, China. Other reagents used in the experiments were
from Sinopharm Chemical Reagent Co., Ltd.
The morphology characterizations of WO3 NSs were performed on a S-3400N scanning
electron microscope (SEM) and a Tecnai G2 F20 S-TWIN transmission electron microscope
(TEM), respectively. A Brucker D8 X-ray diffractometer (XRD) and an ESCALAB 250Xi X-ray
photoelectron spectrometer (XPS) were used for recording XRD and XPS spectra, respectively.
A Unico 4802 UV-Vis spectrophotometer was used to measure absorption spectra.

Preparation of WO3 NSs

WO3 NSs were prepared using a method from literature with some modification [32]. 0.4 g of
Na2WO4·2H2O was dispersed in 300 mL of HNO3 (4.8 M) while stirring. After 10 min of sonicate,
the mixture was subjected to magnetic stirring at room temperature for 72 h. The yellow product
was then gathered after centrifugal separation (8000 rpm, 20 min) and followed by washing with
ultrapure water. After drying in a 50 oC vacuum oven for 12 h, the obtained yellow solid was

Bull. Chem. Soc. Ethiop. 2023, 37(1)

 Spectrophotometric detection of uric acid with enzyme-like reaction 13

stored in a desiccator. Before used, the WO3 NSs were ground into fine powder with an agate
mortar. 50 mg of powder was taken and dispersed into 50 mL ultrapure water following by
sonicate for 2 h.

Detection of H2O2 and UA

Aliquots of 200 μL TMB (10 mM) and 50 μL WO3 NSs (1 mg/mL) were mixed with 200 μL H2O2
with different concentration. 1560 μL HAc-NaAc buffer (0.2 M, pH = 4.5) was then added into
the above solution. After full mixing, it was incubated in a 30 oC water bath for 50 min. Finally,
the absorption spectrum of the resultant solution was recorded.
Both UA and UAO solutions were prepared in Tris buffer (0.1 M, pH = 8.0). The UA detection
was carried out as follows: 100 μL UA with different concentration was incubated with 100 μL
UAO (1 mg/mL) at 37 oC for 25 min to generate H2 O2. Then, 200 μL TMB (10 mM), 50 μL WO3
NSs (1 mg/mL) and 1560 μL HAc–NaAc buffer (0.2 M, pH = 4.5) were added successively. After
fully mixing, it was incubated at 30 oC for another 50 min. Then, the absorption spectrum of the
resultant solution was recorded.
The absorbance at 652 nm was applied to establish the calibration curves for H2O2 and UA.
All experiments were carried out in parallel three times.

Pre-treatment of serum sample

The serum samples from healthy adults were provided by volunteers of our research group and
extracted by Guangxi University Affiliated Hospital (Nanning China). A certain amount of serum
sample was fully mixed with trichloroacetic acid (10%), it was then centrifuged at 5000 rpm for
20 min to remove serum protein. The collected supernatant was diluted with Tris buffer (0.1 M,
pH = 8.0). For standard addition recovery measurement, a certain amount of UA was spiked into
the pretreated sample. The determination of UA was performed according to the procedure
described in Section 1.3.

RESULTS AND DISCUSSION

Composition and morphology of WO3 NSs

The chemical composition and crystal structure of WO3 NSs were identified by XPS and XRD,
respectively. As shown in Figure 1A, the existence of W, O, and C elements can be demonstrated
in the XPS survey spectra of WO3 NSs. The C element was most likely to come from the
absorption of CO2 during the measurement. As can be seen in Figure 1B, the binding energy values
of W4f7/2 and W4f5/2 are observed at 35.23 and 37.15 eV, respectively, which is consistent with
the XPS results of WO3 in the literature [33]. Suggesting the typical characteristic of W6+ oxidation
state. The O1s peak (Figure 1C) at 530.4 eV indicates the existence of O2- in WO3. The other O1s
peaks located at 532.7 eV reveals the presence of adsorbed water molecules on the surface of WO3
[34]. Figure 1D shows the XRD pattern obtained from WO3 NSs powder. Four characteristic
diffraction signals are observed at 16.3°, 24.4°, 25.9°, and 34.4°, which belong to the (001), (110),
(011), and (002) crystal planes of WO3 (JCPDA No.54-0508), respectively. Implying that WO3
existed in its crystalline form. An obvious lamellar structure can be observed in SEM (Figure 1E)
and TEM (Figure 1F) images, indicating that the sample was nanosheets. All these demonstrated
the formation of WO3 NSs.

Bull. Chem. Soc. Ethiop. 2023, 37(1)

14 Jin Yang

Figure 1. Characterization of WO3 NSs: (A). The XPS pattern of WO3 NSs, (B). The XPS
pattern of W 4f (B) and O 1s (C), (D) XRD pattern, (E). SEM image, (F). TEM
image.

Catalytic activity of WO3 NSs

To investigate the catalytic activity of WO3 NSs, TMB and H2O2 were used as chromogenic agent
and oxidant respectively, several control experiments were carried out, and the results are shown
in Figure 2A. The solutions of WO3 NSs, WO3 NSs/TMB and WO3 NSs/H2O2 are all colorless
(inset in Figure 2A) without characteristic absorption peak in the scanning range of 500 to 800
nm (curve a to c). TMB solution involving H2O2 merely shows a pale bluish with a weak
absorption peak (curve d), indicating that TMB was slowly oxidized by H2O2. While TMB
solution involving H2O2 and WO3 NSs results in a deep blue color with a strong absorption peak
at 652 nm (curve e). The observed phenomenon is consistent with the HRP-catalyzed oxidation
of TMB in the presence of H2O2, demonstrating the peroxidase-like catalytic activity of WO3 NSs.

Figure 2. (A) Absorption spectra of solutions with different components. Inset: digital
photos of corresponding solutions. (B) Effect of radical scavenger on the
absorbance of the TMB-H2 O2-WO3 NSs mixture.

Bull. Chem. Soc. Ethiop. 2023, 37(1)

 Spectrophotometric detection of uric acid with enzyme-like reaction 15

Nanomaterials-based peroxidase mimetic, such as MoS2 NSs [26], AuNPs-WS2QDs [35], N-

Fe CDs [36], and carbon quantum dots [37], can promote the decomposition of H2O2 to produce
hydroxyl radical(•OH) intermediates, which further oxidizes TMB to blue oxidation product
(oxTMB). To study the mechanism of WO3 NSs, three •OH scavengers, including isopropyl
alcohol (IPA), methyl alcohol (MA) and L-histidine (His), were added to the TMB-H2O2-WO3
NSs system to evaluate the effects of WO3 NSs on •OH generation. The absorption spectra of
TMB containing H2 O2 and WO3 NSs in the presence and absence of •OH scavengers were
monitored. As shown in Figure 2B, the concentration and type of •OH scavenger have no
significant effect on the absorbance at 652 nm. These results proved that the catalytic activity of
WO3 NSs was not due to the generation of •OH. Similar to Co3 O4-MMT NPs [38] and CoSe2 NFs
[39], whose catalytic activity is achieved by accelerating electron transfer from TMB to H2O2,
rather than by producing •OH. Therefore, the catalytic mechanism of WO3 NSs can be described
as follows. TMB was adsorbed on WO3 NSs surface and provided lone-pair electrons to WO3
NSs, leading to an increase in electrons density of WO3 NSs. Since H2O2 was also adsorbed on
WO3 NSs surface, an increased electron density facilitated the transfer of electrons from TMB to
H2O2. Thus, the oxidation rate of TMB by H2O2 was accelerated.

Figure 3. The steady-state kinetic and catalytic mechanism of WO3 nanosheets. The error
bar represents the standard error obtained from three repeated measurements. (A)
H2O2 concentration is 1.0 mM, TMB concentration is different. (B) TMB
concentration is 1.0 mM, H2O2 concentration is different. (C, D) Double
reciprocal diagram of the catalytic activity of WO3 nanosheets when the
concentration of one substrate (TMB or H2O2) was fixed and the concentration
of the other substrate was changed.

The catalytic performance of WO3 NSs was further studied by steady-state kinetics. As
displayed in Figure 3A and Figure 3B, the oxidation reaction catalyzed by WO3 NSs follows the
typical Michealis-Menten model for both substrates TMB and H2O2. The Michaelis constant (Km),
which is related to the affinity of the enzyme to the substrate, were obtained from the following

Bull. Chem. Soc. Ethiop. 2023, 37(1)

16 Jin Yang

equation: 1/V = (Km/Vmax) x (1/[S]) + 1/Vmax, among them, V, Vmax and [S] represent the initial
reaction velocity, the maximum initial velocity and the substrate concentration, respectively.
According to Figure 3C and Figure 3D, the Km values of WO3 NSs to TMB and H2O2 were
calculated to be 0.733 and 0.164 mM, respectively. It should be noted that a low Km value implies
a high catalytic activity. The Km value of WO3 NSs toward TMB is greater than that of HRP [19]
(0.43 mM), implying that WO3 NSs possesses a lower affinity for TMB than HRP. While the Km
value of WO3 NSs toward H2O2 (3.70 mM) was lower than that of HRP, suggesting that the WO3
NSs exhibits strong affinity for H2O2 [19]. Therefore, as a peroxidase-like enzyme, WO3 NSs can
be applied to the sensitive detection of H2O2 and its related substances.

Applications of WO3 NSs in H2O2 and UA Detection

Similar to other peroxidase mimics, the catalytic activity of WO3 NSs was also affected by
external factors. To achieve the best response, we studied the influences of experimental
conditions including pH value, TMB concentration, and incubation time to optimize the H2O2-
mediated TMB chromogenic reaction in a 30 oC water bath. As shown in Figure 4, the favorable
pH, TMB concentration, and incubation time for the WO3 NSs-catalyzed TMB oxidation are 4.5,
1.0 mM, and 50 min, respectively. Under the above experimental conditions, a simple method for
colorimetric detection of H2O2 was established. As displayed in Figure 5A, the absorbance
increases with the increase of H2O2 concentration, and the obvious change of solution color can
be observed by the naked eye (Figure 5A inset). Furthermore, the absorbance was linear to H2O2
concentration in the range of 2.0-180 μM (Figure 5B), the regression equation could be defined
as A = 0.0043C (μM) + 0.023 (R2 = 0.991). The limit of detection (LOD) at 3δ/k (where δ is the
standard deviation of 11 blank solution measurements, and k is the slope of calibration curve) for
H2O2 was 1.34 μM, suggesting high sensitivity for H2O2 detection.

Figure 4. Effect of (A) pH value, (B) TMB concentration and (C) reaction time on the
catalytic activity of the WO3 NSs.

Under the catalysis of UAO, UA is oxidized to produce H2O2 quantitatively. Thus, a method
for the determination of UA was further developed. As shown in Figure 5C, with the increase of
UA concentration, the absorbance at 652 nm increases gradually, accompanied with obvious color
change from colorless to light blue and dark blue (Figure 5C inset), indicating the possibility of
visual detection of UA. As can be seen in Figure 5D, the absorbance exhibits a linear response to
UA concentration over the range of 2.0 - 180 μM, the calibration curve can be depicted as A
=0.0046C (μM) + 0.0198 (R2 = 0.996). On the basis of 3δ/k, the LOD for UA was 1.25 μM, which
is much lower than the lowest concentration of UA in human serum (0.12 mM) [4, 5], implying a
high sensitivity method for UA detection. The relative standard deviation (RSD) was 1.1% for
determining 40.0 μM UA (n = 3), indicating a good precision of the method. As summarized in
Table 1, the developed method is superior to most of the approaches for UA detection in
sensitivity or linear range.

Bull. Chem. Soc. Ethiop. 2023, 37(1)

 Spectrophotometric detection of uric acid with enzyme-like reaction 17

Figure 5. (A)Absorption spectra of TMB-WO3 NSs mixture with various concentrations

of H2O2. Inset: digital photos of corresponding solutions. (B) The linear relation
of absorbance with H2O2 concentration. (C) Absorption spectra of TMB-WO3
NSs mixture with various concentrations of UA. Inset: digital photos of
corresponding solutions. (D) The linear relation of absorbance with UA
concentration.

Table 1. Comparison with some reported methods for the detection of uric acid.

Linear
Materials Detection Real Recovery
Method range Reference
used limit (μM) sample (%)
(μM)
Fe3O4@fatty
Colorimetric 2.8 5-250 Serum 94.7-103.4 [40]
acid MNPs
Pt@Ag NFs Colorimetric 0.3 0.5-150 Serum 96.8-103.3 [41]
Ag2V4O11
Colorimetric 0.35 1-110 * * [42]
NBs
Au NPs Colorimetric 0.04 0.1-30 Urine 96.1-103.1 [43]
Fe@NCDs Colorimetric 0.64 2-150 Urine 92.0-103.4 [44]
CoP NSs Colorimetric 1.0 1-200 Urine 99.6-106.5 [45]
WO3 NSs Colorimetric 1.25 2-180 Serum 92.6-105.5 This word

The potential interferences including Glu, Cys, Gly, His, Ser, Thr, Na+, K+, and Mg2+, which
might coexist in serum and interfere with the determination of UA, were chosen to assess the
selectivity of the method. The concentrations of UA and interferents were 0.1 and 1.0 mM,
respectively. As displayed in Figure 6, the absorbance of the solution containing interference is
far lower than that of UA because of the high selectivity of UAO to UA, indicating good
selectivity for UA determination.

Bull. Chem. Soc. Ethiop. 2023, 37(1)

18 Jin Yang

Figure 6. Selectivity evaluation for UA determination. The concentrations of UA and

interferents were 0.1 and 1.0 mM respectively. (a. Glu; b. Cys; c. Gly; d. His; e.
Ser; f. Thr; g. Na+ ; h. K+; i. K+; j. Mg2+; k. UA).

To evaluate the feasibility of the method in practical application, the concentration of UA in

human serum was determined, and a standard addition method was used to verify the accuracy of
the method. The results are displayed in Table 2. Considering the sample dilution (37 times)
caused by the determination process, the concentration of UA in the original serum samples are
0.26 and 0.19 mM, respectively, which are within the normal range (0.12 to 0.46 mM). The
recoveries of UA in serum samples ranged from 92.6% to 105.5%, and the RSD at each level was
less than 3.3%, confirming an accurate and reliable method.

Table 2. Determination results of UA in human serum samples (n = 3).

Sample Added (μM) Found (μM) Recovery (%) RSD (%)

0 7.092 0 1.5
1 20 27.65 102.8 1.0
40 44.13 92.60 3.3
0 5.241 0 2.3
2 40 47.46 105.5 2.7
80 83.39 97.68 2.5

CONCLUSIONS
WO3 NSs was prepared at room temperature, which exhibited peroxidase-like activity and could
catalyze H2O2 to oxidize colorless TMB into blue oxTMB. Taking the advantages of the catalytic
activity of WO3 NSs and the specificity of UAO, a sensitive and selective spectrophotometric
method for UA detection was developed. A good linear relationship between absorbance and UA
concentration over the range of 2.0–180 μM was achieved with LOD of 1.25 μM. More
importantly, the method has been used for determining UA in serum with good accuracy and
precision, showing its promising potential applications.

ACKNOWLEDGEMENTS
This work was supported by the Start-up Fund for Talent Introduction of Guangxi University
(A3040051025) and the Innovation Project of Guangxi Graduate Education (No.
YCSW2021050).
Bull. Chem. Soc. Ethiop. 2023, 37(1)
 Spectrophotometric detection of uric acid with enzyme-like reaction 19

REFERENCES

1. Huang, Z.; Xie, N.; Illes, P.; Di Virgilio, F.; Ulrich, H.; Semyanov, A.; Verkhratsky, A.;
Sperlagh, B.; Yu, S.-G.; Huang, C.; Tang, Y. From purines to purinergic signalling: Molecular
functions and human diseases. Signal Transduct. Target Ther. 2021, 6, 162.
2. Yang, J.; Huang, Z.; Hu, Y.; Ge, J.; Li, J.; Li, Z. A facile fluorescence assay for rapid and
sensitive detection of uric acid based on carbon dots and MnO2 nanosheets. New J. Chem.
2018, 42, 15121-15126.
3. Virdis, A.; Masi, S.; Casiglia, E.; Tikhonoff, V.; Cicero, A.F.G.; Ungar, A.; Rivasi, G.;
Salvetti, M.; Barbagallo, C.M.; Bombelli, M.; Dell’Oro, R.; Bruno, B.; Lippa, L.; D’Elia, L.;
Verdecchia, P.; Mallamaci, F.; Cirillo, M.; Rattazzi, M.; Cirillo, P.; Gesualdo, L.; Mazza, A.;
Giannattasio, C.; Maloberti, A.; Volpe, M.; Tocci, G.; Georgiopoulos, G.; Iaccarino, G.;
Nazzaro, P.; Parati, G.; Palatini, P.; Galletti, F.; Ferri, C.; Desideri, G.; Viazzi, F.; Pontremoli,
R.; Muiesan, M.L.; Grassi, G.; Borghi, C. null, n., Identification of the uric acid thresholds
predicting an increased total and cardiovascular mortality over 20 years. Hypertension 2020,
75, 302-308.
4. Yang, Y.; Song, Y.; Bo, X.; Min, J.; Pak, O. S.; Zhu, L.; Wang, M.; Tu, J.; Kogan, A.; Zhang,
H.; Hsiai, T. K.; Li, Z.; Gao, W. A laser-engraved wearable sensor for sensitive detection of
uric acid and tyrosine in sweat. Nat. Biotechnol. 2020, 38, 217-224.
5. Wang, Z.; Guo, H.; Gui, R.; Jin, H.; Xia, J.; Zhang, F. Simultaneous and selective
measurement of dopamine and uric acid using glassy carbon electrodes modified with a
complex of gold nanoparticles and multiwall carbon nanotubes. Sen. Actuat. B Chem. 2018,
255, 2069-2077.
6. Ahmad, M.; Faraazi, A.; Aamir, N. The effect of Ocimum sanctum and ledum palustre on
serum uric acid level in patients suffering from gouty arthritis and hyperuricaemia. Bull.
Chem. Soc. Ethiop. 2013, 27, 469-473.
7. Wang, X.-y.; Zhu, G.-b.; Cao, W.-d.; Liu, Z.-j.; Pan, C.-g.; Hu, W.-j.; Zhao, W.-y.; Sun, J.-f.
A novel ratiometric fluorescent probe for the detection of uric acid in human blood based on
H2O2-mediated fluorescence quenching of gold/silver nanoclusters. Talanta 2019, 191, 46-53.
8. Meng, F.; Yin, H.; Li, Y.; Zheng, S.; Gan, F.; Ye, G. One-step synthesis of enzyme-stabilized
gold nanoclusters for fluorescent ratiometric detection of hydrogen peroxide, glucose and uric
acid. Microchem. J. 2018, 141, 431-437.
9. Motshakeri, M.; Phillips, A.R.J.; Kilmartin, P.A. Application of cyclic voltammetry to analyse
uric acid and reducing agents in commercial milks. Food Chem. 2019, 293, 23-31.
10. Chen, Y.; Ji, P.; Ma, G.; Song, Z.; Tang, B. Q.; Li, T. Simultaneous determination of cellular
adenosine nucleotides, malondialdehyde, and uric acid using HPLC. Biomed. Chromatogr.
2021, 35, e5156.
11. Yang, M.; Wang, H.; Liu, P.; Cheng, J. A 3D electrochemical biosensor based on super-
aligned carbon nanotube array for point-of-care uric acid monitoring. Biosens. Bioelectron.
2021, 179, 113082.
12. Wang, X.; Chen, S.; Tang, X.; Lin, D.; Qiu, P. Ultrasensitive detection of uric acid in serum
of patients with gout by a new assay based on Pt@Ag nanoflowers. RSC Adv. 2019, 9, 36578-
36585.
13. Altinkaynak, C.; Turk, M.; Ekremoglu, M.; Ozdemir, N. Peroxidase-like activity of
hemoglobin-based hybrid materials against different substrates and their enhanced application
for H2O2 detection. Bull. Chem. Soc. Ethiop. 2021, 35, 537-550.
14. Wang, L.; Yu, L.; Ge, H.; Bu, Y.; Sun, M.; Huang, D.; Wang, S. A novel reversible dual-
mode probe based on amorphous carbon nanodots for the detection of mercury ion and
glutathione. Microchem. J. 2022, 175, 107181.
15. Nie, Q.; Cai, Q.; Xu, H.; Qiao, Z.; Li, Z. A facile colorimetric method for highly sensitive
ascorbic acid detection by using CoOOH nanosheets. Anal. Methods 2018, 10, 2623-2628.

Bull. Chem. Soc. Ethiop. 2023, 37(1)

20 Jin Yang

16. Chen, C.; Zhang, S.; Zhang, C.; Li, L.; Zhu, J.; Liu, J. TMB-assembly as nanosubstrate
construction colorimetric kit for highly sensitive and selective detection of H2O2 and
monoamine oxidase-A based on Fenton reaction. Microchem. J. 2019, 150, 104177.
17. Yang, C.; Zhang, M.; Wang, W.; Wang, Y.; Tang, J. UV-Vis detection of hydrogen peroxide
using horseradish peroxidase/copper phosphate hybrid nanoflowers. Enzyme Microb.
Technol. 2020, 140, 109620.
18. Ocsoy, I.; Dogru, E.; Usta, S. A new generation of flowerlike horseradish peroxides as a
nanobiocatalyst for superior enzymatic activity. Enzyme Microb. Technol. 2015, 75-76, 25-
29.
19. Gao, L.; Zhuang, J.; Nie, L.; Zhang, J.; Zhang, Y.; Gu, N.; Wang, T.; Feng, J.; Yang, D.;
Perrett, S.; Yan, X. Intrinsic peroxidase-like activity of ferromagnetic nanoparticles. Nat.
Nanotechnol. 2007, 2, 577-583.
20. Qin, X.; Yuan, C.; Shi, R.; Wang, Y. A double signal optical probe composed of carbon
quantum dots and Au@Ag nanoparticles grown in situ for the high sensitivity detection of
ellagic acid. J. Mol. Liq. 2021, 323, 114594.
21. Bagheri, N.; Khataee, A.; Habibi, B.; Hassanzadeh, J. Mimetic Ag nanoparticle/Zn-based
MOF nanocomposite (AgNPs@ZnMOF) capped with molecularly imprinted polymer for the
selective detection of patulin. Talanta 2018, 179, 710-718.
22. Xi, Z.; Wei, K.; Wang, Q.; Kim, M. J.; Sun, S.; Fung, V.; Xia, X. Nickel–platinum
nanoparticles as peroxidase mimics with a record high catalytic efficiency. J. Am. Chem. Soc.
2021, 143, 2660-2664.
23. Ge, J.; Xing, K.; Geng, X.; Hu, Y.; Shen, X.; Zhang, L.; Li, Z. Human serum albumin
templated MnO2 nanosheets are oxidase mimics for colorimetric determination of hydrogen
peroxide and for enzymatic determination of glucose. Microchim. Acta 2018, 185, 559.
24. Sun, J.; Li, L.; Kong, Q.; Zhang, Y.; Zhao, P.; Ge, S.; Cui, K.; Yu, J. Mimic peroxidase-
transfer enhancement of photoelectrochemical aptasensing via CuO nanoflowers
functionalized lab-on-paper device with a controllable fluid separator. Biosens. Bioelectron.
2019, 133, 32-38.
25. Jin, X.; Yin, W.; Ni, G.; Peng, J. Hydrogen-bonding-induced colorimetric detection of
melamine based on the peroxidase activity of gelatin-coated cerium oxide nanospheres. Anal.
Methods. 2018, 10, 841-847.
26. Shi, R.; He, Q.; Cheng, S.; Chen, B.; Wang, Y. Determination of glucose by using MoS2
nanosheets as a peroxidase mimetic enzyme. New J. Chem. 2021, 45, 18048-18053.
27. Yuan, C.; Qin, X.; Xu, Y.; Jing, Q.; Shi, R.; Wang, Y. High sensitivity detection of H2O2 and
glucose based on carbon quantum dots-catalyzed 3,3′,5,5′-tetramethylbenzidine oxidation.
Microchem. J. 2020, 159, 105365.
28. Zhu, Q.; Mao, H.; Li, J.; Hua, J.; Wang, J.; Yang, R.; Li, Z. A glycine-functionalized graphene
quantum dots synthesized by a facile post-modification strategy for a sensitive and selective
fluorescence sensor of mercury ions. Spectrochim. Acta A Mol. Biomol. Spectrosc. 2021, 247,
119090.
29. Ma, Y.; Zhao, M.; Cai, B.; Wang, W.; Ye, Z.; Huang, J. 3D graphene network@WO3
nanowire composites: A multifunctional colorimetric and electrochemical biosensing
platform. Chem. Commun. 2014, 50, 11135-11138.
30. Liu, Z.; Gong, S.; Wang, Y.; Chen, T.; Niu, Y.; Xu, Y. Recognition of the enzymatically active
and inhibitive oxygenous groups on WO3–x quantum dots by chemical deactivation and
density functional theory calculations. ACS Appl. Bio Mater. 2020, 3, 1459-1468.
31. Li, Z.; Liu, X.; Liang, X.-H.; Zhong, J.; Guo, L.; Fu, F. Colorimetric determination of xanthine
in urine based on peroxidase-like activity of WO3 nanosheets. Talanta 2019, 204, 278-284.
32. Liang, L.; Li, K.; Xiao, C.; Fan, S.; Liu, J.; Zhang, W.; Xu, W.; Tong, W.; Liao, J.; Zhou, Y.;
Ye, B.; Xie, Y. Vacancy associates-rich ultrathin nanosheets for high performance and flexible
nonvolatile memory device. J. Am. Chem. Soc. 2015, 137, 3102-3108.

Bull. Chem. Soc. Ethiop. 2023, 37(1)

 Spectrophotometric detection of uric acid with enzyme-like reaction 21

33. Guo, Q.; Zhao, X.; Li, Z.; Wang, D.; Nie, G. A novel solid-state electrochromic supercapacitor
with high energy storage capacity and cycle stability based on poly(5-formylindole)/WO3
honeycombed porous nanocomposites. Chem. Eng. J. 2020, 384, 123370.
34. Sharma, S.; Basu, S. Highly reusable visible light active hierarchical porous WO3/SiO2
monolith in centimeter length scale for enhanced photocatalytic degradation of toxic
pollutants. Sep. Purif. Technol. 2020, 231, 115916.
35. Vinita; Nirala, N.R.; Prakash, R. Facile and selective colorimetric assay of choline based on
AuNPs-WS2QDs as a peroxidase mimic. Microchem. J. 2021, 167, 106312.
36. Yue, G.; Li, S.; Liu, W.; Ding, F.; Zou, P.; Wang, X.; Zhao, Q.; Rao, H. Ratiometric
fluorescence based on silver clusters and N, Fe doped carbon dots for determination of H2O2
and UA: N, Fe doped carbon dots as mimetic peroxidase. Sen. Actuat. B Chem. 2019, 287,
408-415.
37. Shu X.; Wang D.; Yuan C.; Qin X.; Wang Y. Colorimetric determination of sarcosine with
carbon quantum dots as mimetic peroxidase. Chem. J. Chinese Universities 2021, 42, 1761-
1767.
38. Zhu, X.; Chen, W.; Wu, K.; Li, H.; Fu, M.; Liu, Q.; Zhang, X. A colorimetric sensor of H2O2
based on Co3O4–montmorillonite nanocomposites with peroxidase activity. New J. Chem.
2018, 42, 1501-1509.
39. Warkhade, S.K.; Singh, R.P.; Das, R.S.; Gaikwad, G.S.; Zodape, S.P.; Pratap, U.R.;
Maldhure, A.; Wankhade, A.V. CoSe2 nanoflakes: An artificial nanoenzyme with excellent
peroxidase like activity. Inorg. Chem. Commun. 2021, 126, 108461.
40. Wen, J.; Yun, Z.; Zhili, C.; Yang, Y. Peroxidase-like activity of Fe3O4@fatty acid-
nanoparticles and their application for the detection of uric acid. New J. Chem. 2020, 44,
18608-18615.
41. Wang, X.; Chen, S.; Tang, X.; Lin, D.; Qiu, P. Ultrasensitive detection of uric acid in serum
of patients with gout by a new assay based on Pt@Ag nanoflowers. RSC Adv. 2019, 9, 36578-
36585.
42. Sun, L.; Shen, H.; Zheng, L.; Gao, P.; Xiang, Z. Colorimetric detection of uric acid based on
peroxidase-like activity of Ag2 V4O11 nanobelts. J. Electron. Mater. 2021, 50, 3907-3915.
43. Li, F.; He, T.; Wu, S.; Peng, Z.; Qiu, P.; Tang, X. Visual and colorimetric detection of uric
acid in human serum and urine using chitosan stabilized gold nanoparticles. Microchem. J.
2021, 164, 105987.
44. Liang, C.; Lan, Y.; Sun, Z.; Zhou, L.; Li, Y.; Liang, X.; Qin, X. Synthesis of carbon quantum
dots with iron and nitrogen from Passiflora edulis and their peroxidase-mimicking activity
for colorimetric determination of uric acid. Microchim. Acta 2020, 187, 405.
45. He, Y.; Qi, F.; Niu, X.; Zhang, W.; Zhang, X.; Pan, J. Uricase-free on-demand colorimetric
biosensing of uric acid enabled by integrated CoP nanosheet arrays as a monolithic peroxidase
mimic. Anal. Chim. Acta 2018, 1021, 113-120.

Bull. Chem. Soc. Ethiop. 2023, 37(1)