WO2015047079A2 - Application of enzyme-quantum dots hybrid system for the determination of uric acid - Google Patents

Application of enzyme-quantum dots hybrid system for the determination of uric acid Download PDF

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WO2015047079A2
WO2015047079A2 PCT/MY2014/000256 MY2014000256W WO2015047079A2 WO 2015047079 A2 WO2015047079 A2 WO 2015047079A2 MY 2014000256 W MY2014000256 W MY 2014000256W WO 2015047079 A2 WO2015047079 A2 WO 2015047079A2
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uric acid
determination
hybrid system
qds
quantum dots
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WO2015047079A3 (en
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Nur Ellina AZMI
Noor Izaanin RAMLI
Hamidah SIDEK
Samsulida ABD RAHMAN
Nurhayati ARIFFIN
Jaafar ABDULLAH
Muhammad Azmi ABDUL HAMID
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Sirim Berhad
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/588—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with semiconductor nanocrystal label, e.g. quantum dots
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0044—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on other nitrogen compounds as donors (1.7)
    • C12N9/0046—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on other nitrogen compounds as donors (1.7) with oxygen as acceptor (1.7.3)
    • C12N9/0048—Uricase (1.7.3.3)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/62—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving uric acid

Definitions

  • the present invention relates generally to quantum dots for the determination of uric acid and more particularly to determination of uric acid through fluorescence quenching of negatively charged quantum dots induced by hydrogen peroxide produced by the enzymatic reaction in the hybrid uricase/HRP-CdS QDs system.
  • Table 1 shows evaluation of the potential interference in the sample
  • Figure 3 shows TEM of MP A capped CdS QDs.
  • Figure 5 shows effect of enzyme ratio (uricase:HRP) on the uric acid measurement with the concentration of uric acid at 0.25 mM.

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  • General Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

The present invention relates to a uricase/HRP-CdS QDs hydrid system for the determination of uric acid. The QDs was used as an electron donor, whereas uricase and HRP (horseradish peroxidase) were used as acceptors for the oxidation/reduction reactions involved in oxidizing uric acid to allaintoin and hydrogen peroxide. The hydrogen peroxide produced was able to quench the QDs fluorescence which was proportional to uric acid concentration. The hybrid system demonstrated maximum activity of uricase/HRP at ratio of 1/1 and pH 7.0, respectively. The linearity of the hybrid system towards uric acid was in the concentration range of 125-1000 μΜ with detection limit of 125 μΜ.

Description

APPLICATION OF ENZYME-QUANTUM DOTS HYBRID SYSTEM FOR THE
DETERMINATION OF URIC ACID
Technical Field of the Invention
The present invention relates generally to quantum dots for the determination of uric acid and more particularly to determination of uric acid through fluorescence quenching of negatively charged quantum dots induced by hydrogen peroxide produced by the enzymatic reaction in the hybrid uricase/HRP-CdS QDs system.
Background of the Invention The development of a wide spectrum of nanoscale technologies is beginning to change the foundations of disease diagnosis, treatment, and prevention^ Quantum dots (QDs) are one of nanomaterials that have gain much attention on research in many fields such as physical, chemical and biological sciences. The unique optoelectronic properties of these particles, particularly in terms of brightness, photostability, narrow tunable emission, and broad absorption, are attractive in many applications compared to organic fluorophores [CP. Huang, Y.K. Li, T.M. Chen, Biosens. Bioelectron. 22 (2007)]. One area of application are imaging with QDs [J. Pan, S.S. Feng, Biomaterials 30 (2009)], where the optical properties are advantageous in comparison to most organic fluorophores. Another growing area is the application in the development of biosensors. For example, antibodies were conjugated to QDs for use in sandwich immunoassays [M. Sun, L. Du, S. Gao, Y. Bao, S. Wang, Steroid 75 (2010)], and in fluorescence resonance energy transfer (FRET) based sensors for the detection of 2,4,6- trinitrotoluene (TNT) [Y.Q. Wang, W.S. Zou, Talanta 85 (2011)]. Recently, the hybrid systems containing QDs coupled with various bio-molecules stimulate growing interests in the research areas of biotechnology and nanotechnology. Through the bioconjugation of QDs, hybrid materials have demonstrated both the unique optical properties of QDs and high specificities toward biomolecules, such as oligonucleotides and proteins [Z. Kaul et al. Cell Res. 13 (2003)]. Several papers had reported QDs-enzyme nanohybrids for biosensor applications [K.E. Sapsford et al. Sensors 6 (2006)]. The QDs offer both as a fluorophore and as a multifunctional nano- scaffold for the attachment of biomolecules or other moieties due to its fruitful surface chemistry as well as large surface area [W. Vastarella, R. Nicastri, Talanta 66 (2005)].
Uric acid (2,6,8-trihydroxypurine, UA) is end product of purine metabolism in human system [D. Martinez-Perez, M.L. Ferrer, C.R. Mate, Anal. Biochem. 322 (2003)]. The assay of UA in body fluids (e.g. serum and urine) is an important marker molecule for diseases related with variations of plasma urate level such as hyperuricemia (gout), renal impairment, leukemia, ketoacidosis, cardiovascular disease and lactate excess [F.F. Zhang, et al., Talanta 68 (2006)]. The normal level of UA in serum is in the range of 0.13-0.46 mM (2.18-7.7 mg/dL) [C.R. Raj, T.J. Ohsaka, J. Electroanal. Chem. 540
(2003) ] and 1.49-4.46 mM (25-74 mg/dL) [R.C. Matos, el al., Analyst 125 (2000)] in urinary excreation.
Thus, the determination of uric acid concentration is an urgent requirement for clinical analysis for disease diagnosis. Various analytical methods for detecting uric acid have been reported such as spectrophotometry [S.H. Huang, et al., Biosens. Bioelectron. 19
(2004) ], electrochemical techniques [C.R. Raj, T.J. Ohsaka, J. Electroanal. Chem. 540 (2003)], flow-injection chemiluminescence (FI-CL) [M. Tabata, et al, Appl. Biochem. 6 (1984)], high performance liquid chromatographic (HLPC) [J. Wang, et al., Anal. Chem. 59 (1987)] and fluorescence method [J. Galban, et al., Talanta 54 (2001)], etc. However, these methods involve laborious, slow procedures, expensive reagents and also associated with selectivity and sensitivity. Therefore, it is necessary to develop simple, accurate and sensitive methods for the determination of uric acid. In this present invention, it was found that the use of QDs for the determination of uric acid through fluorescence quenching of negatively charged QDs induced by hydrogen peroxide produced by the enzymatic reaction in the hybrid uricase/HRP-CdS QDs system. The system also provides a promising tool for clinical diagnosis of uric acid and other fields of medical applications.
Summary of the Invention
Accordingly, it is a primary object of the present invention to provide a simple, accurate and sensitive method for the determination of uric acid.
Therefore the first aspect of the present invention can be accomplished by providing a hybrid system for the determination of uric acid comprising:
uricase and horseradish peroxidase enzyme ratio having specific reaction time; quantum dots as optical fluorescent indicator; and
a buffer solution for optimum pH value for hydrogen peroxide quenching effects of uric acid on the quantum dots fluorescence intensity.
Brief Description of the Drawings
The present invention can be more fully understood by referring to the accompany tables and figures, in which:
Table 1 shows evaluation of the potential interference in the sample
Table 2 shows determination of uric acid in urine samples using the developed enzyme- QDs hybrid system and uric acid assay kit Figure 1 shows illustration of the hybrid uricase H P based on H202-sensitive QDs. Figure 2 shows fluorescence emission spectra of MP A capped CdS QDs.
Figure 3 shows TEM of MP A capped CdS QDs.
Figure 4 show effect of pH on the fluorescence properties of uricase/HRP-CdS QDs in buffer containing 0.25 mM uric acid.
Figure 5 shows effect of enzyme ratio (uricase:HRP) on the uric acid measurement with the concentration of uric acid at 0.25 mM.
Figure 6 shows quenc ng effects of QDs towards different concentration of uric acid.
Detailed Description of the Invention
EXPERIMENTAL
Reagents
Cadmium chloride hydrate (CdCl2) (98%) and 3-Mercaptopropionic acid (> 99%) were purchased from Aldrich. Uric acid (99%), uricase from Bacillus Fastidiosus lyophilized, peroxidase from horseradish Type IV and sodium sulfide hydrate (60%) were obtained from Sigma. Sodium hydroxide was purchased from Scharlau. Phosphate buffer was prepared by mixing both sodium dihydrogen phosphate anhydrous (99%) (Scharlau) and di-sodium hydrogen phosphate (98%) (HmbG Chemicals). . Other reagents were analytical grade and used without further purification. Water used for preparation of aqueous solution was purified using Millipore Milli-Q water purification system (18.2 ΜΩ-cm). Preparation of CdS QDs
The synthetic procedure of mercaptopropionic acid (MPA)-capped cadmium sulfide (CdS) QDs was based on previous published work with slight modification [M. Koneswaran, R. Narayanaswamy, Sensors and Actuators B 139 (2009)] is as follows: 0.5 mmol MPA and cadmium chloride (CdCl2) were dissolved in 250 mL of double distilled water. Then pH 6 was obtained by adding NaOH under constant stirring followed by N2 purging for 1 hour. Then, Na2S (0.5 mmol) was added dropwise under N2 environment. Finally, the solution was left for 24 hours under constant stirring and room temperature.
Instrumentation
UY-Vis absorption spectra were recorded using spectrophotometer Varian-Cary win UV 50. While emission spectra were recorded by spectrofluorescence Tecan Infinite M200 and spectrometer fluorescence Ocean Optics with mercury light source (EFOS Novacure spectrophotometer).
Procedure for determination of uric acid
Uric acid (400 μΐ,), uricase (22 μΐ,) and horseradish peroxidase (HRP) (8 μϋ) were mixed in phosphate buffer solution (PBS) pH 7. Then, the mixture was incubated for 2 minutes before adding CdS QDs solution (500 μΐ,). The fluorescence spectra of the mixture were determined in the emission wavelength range of 450-800 nm. The fluorescence intensity of the mixture was recorded at wavelength of 565 nm. For real sample analysis, several urine samples were collected from various volunteers. The samples were centrifuged at 1000 rpm for 5 min to remove any impurity. Then the samples were kept in aliquots in eppendorf tube and stored at - 80 °C for further use.
Detection of uric acid using assay kit (Cayman)
100 μΐ^ of diluted assay buffer (tris HC1 7.5) was mixed with 20 μΐ, of fluorometric detector (10-acetyl-3,7-dmydroxyphenoxazine) and 10 μΐ, of the urine sample which has been diluted for 10 times in 96- well plates. Then the reaction was initiated by adding 20 iL of enzyme mixture. The plate was then incubated for 15 min at room temperature. The assay was performed using Tecan fluorescence spectrophotometer with excitation wavelength of 535 nm and emission wavelength of 590 nm.
RESULTS AND DISCUSSIONS
Characterization of MP A capped CdS QDs
Functionalized MPA-capped CdS QDs was optically characterized by using UV-vis absorption spectroscopy and fluorometry. The absorption of MPA-capped CdS QDs was obtained at wavelength of 406 nm. The fluorescence emission peak was observed at wavelength of 565 nm with excitation of 406 nm (Figure 2). The morphology of functionalized MPA-capped CdS QDs was also studied by transmission electron microscopy (TEM). The TEM image of CdS shows spherical shape, dispersed with diameter ranging from 8-15 nm (Figure 3).
Principle of detection
The basic principle of QDs-uric acid detection is illustrated in Figure 1. The detection was based on optical technique which uses the combination of the uricase reaction and the quenching effect of hydrogen peroxide (H202) produced on the QDs fluorescence emission. In the presence of uricase, uric acid is oxidized and H202 is derived from deoxidation of 02. Based on the quenching effect of H202 on the QDs, the uric acid is detected via momtoring the change of the QDs fluorescence. Usually, the quenching of fluorescence emission of QDs may happen by energy transfer [J. Tang, R.A. Marcus, The J. Chem. Physics 125 (2006)], charge diverting [X. Ji, J. Zheng, et al., The J. Physical Chem. B 109 (2005)], and surface absorption [C. Dong, et al., The J. Physical Chem. B 110 (2006)], which could change the surface state of QDs. In this study, the CdS QDs capped MPA was used as indicator. Upon addition of uric acid to the mixture containing hybrid uricase/HRP-CdS QDs, uric acid will be oxidized by oxygen under enzymatic reaction to yield allaintoin, C02 and H202. In the presence of H202, it is assumed that the electron-transfer reaction occurred at the surface of the QDs where H202 was reduced to oxygen (02), which in turn lied in electron/hole traps on the QDs and could be used as a good electron donor, thus forrning the non-fluorescent QDs anion and leading to reduce the fluorescence [L.H. Cao, J. et al., Chem. Eur. J. 14 (2008)]. Another reason may be due to the thiol groups of MPA tagged on the surface of the QDs through Cd-S bonding are readily oxidized to form an organic disulfide product (RS-SR), which causing more MPA molecules are detached from the surface of the QDs, thus quenching the fluorescence of the QDs [Y.C. Shiang, C.C. Huang, H.T. Chang, Chem. Commun. 14 (2009)].
Effect of pH on the hybrid uricase/HRP-CdS system
Several detection conditions were optimized to achieve sensitive hybrid system of uricase/HRP-CdS for uric acid determination. Since the pH value of the reaction solution has a significant influence on the response of uricase HRP-CdS system, the quenching effects of uric acid on the QDs fluorescence intensity was studied over the pH range from 5.0 to 9.0. As illustrated in Figure 4, the optimum pH value was observed at pH 7 which is nearly similar reported by Zhang et al. Analytica Chimica Acta 519 (2004) which use phosphate buffer pH 6.9 and is in good agreement with reported for soluble uricase. The low fluorescence intensity in acidic medium is the result of dissociation of the nanoparticles due to protonation of the surface binding thiolates [M. Gao, el at., The J. Physical Chem. B 102 (1998)]. When the pH increased, the deprotonation of the thiol group in the mercaptopropionic acid molecule occurs. This deprotonation could strengthen the covalent bond between Cd and mercaptopropionic acid molecule, which bring about the fluorescence intensity enhance with pH increasing. However, the fluorescence intensity begins to decrease with the further increase of pH value. Therefore, the pH value of 7 was selected for further studies.
Effect of enzyme ratio on the fluorescence intensity
To achieve sensitive detection of uric acid, effect of different enzyme ratio on hybrid uricase/HRP-CdS QDs system have been investigated. Five different ratio uricase: HRP of 2U:2U, 5U:5U, 10U: 10U, 20U: 10U and 10U:20U were used in this study. As shown in Figure 5, the fluorescence intensity increased as increasing of enzyme unit. The reaction time reached saturation after 30 minutes of reaction. Although the ratio of uricase:HRP of 20U: 10U gave the highest intensity changed but for further experiment ratio 5U:5U was selected because it was sufficient for the reaction to occur in the system. The system was similar as reported before [D. Martinez-Perez, M.L. Ferrer, C.R. Mateo, Analytical Biochemistry 322 (2003)] which use low concentration of 1 : 1 uricase:HRP enzyme ratio for uric acid detection. Therefore, the ratio of uricase:HRP of 5U:5U and reaction time of 30 min was selected for further studies.
Analytical performance of the hybrid uricase HRP-CdS QDs towards uric acid
Figure 6 shows the dynamic response of the hybrid uricase HRP-CdS QDs system obtained by addition of uric acid concentrations ranging from 0.0-1.0 mM. The decreased of the fluorescence intensity produced due to the quenching effect of H2O2 on the QDs which is proportional to the concentrations of uric acid. A linear response of the system was obtained at uric acid concentration range of 125-1000 μΜ (slope = 37239, R2 = 0.992) with the detection limit calculated to be at 125 μΜ.
Evaluation of potential interference in the sample
There are commonly existing foreign substances which may affect the performance of the detection system. In order to assess the precision of the proposed method, solutions containing different potential interference are tested under the same condition in the existing of 250 μΜ uric acid. Table 1, shows the change of fluorescence intensity in the presence of interfering substances. The calculated RSD value were within 100 ±5% which indicate that urea, ascorbic acid, glucose, K+, Na+ and NH4 + did not show any significant interference in this system. Therefore this method may be applied for the direct determination of uric acid in urine sample. 6
Table 1. Evaluation of the potential interference in the sample
Figure imgf000010_0001
Real sample analysis
To investigate the feasibility of the developed enzyme-QDs hybrid system for analysis of uric acid in biological fluids, uric acid in human urine samples were measured and compared with those obtained from assay kit. The results from both methods are summarized in Table 2. The relative error between the developed enzyme-QDs hybrid system and assay kit method for the determination of uric acid in the urine samples were about 4-6 %. Statistical analysis for comparing the two means of the developed enzyme- QDs hybrid system and the assay kit was also evaluated. The statistical analysis was carried out utilizing the method described by Miller and Miller [J.N. Miller and J.C. Miller, Statistics and Chemometrics for analytical chemistry, 4th Edition, Pearson Education Limited, Essex, England, 2000]. As shown in Table 2, since the calculated values of 11 | are less than the critical value, the difference between the two methods is insignificant at the 95% confident level and the null hypothesis is accepted. This result shows that the two methods used for the determination of uric acid in urine samples were in good agreement and were comparable. Table 2. Determination of uric acid in urine samples using the developed enzyme-QDs hybrid system and uric acid assay kit
Urine sample Hybrid system Assay kit Calculated
(mM) (mM) t-test
(n=3) (n=3)
1 4.12 3.94 0.35
2 2.99 2.82 0.15
3 2.66 2.49 1.86
4 0.64 0.49 1.84
' 5 1.96 1.95 0.11
Note: the critical value, t4
CONCLUSIONS In summary, a novel and convenient technique for uric acid determination based on the quenching of the fluorescence of CdS QDs has been developed. The possible quenching mechanism is due to the H202 produced from the uricase-catalyzed oxidation of uric acid. Under optimum conditions, the method achieved a good linear relationship between relative fluorescence intensity of the system and concentration of uric acid in the range from 125-1000 μΜ. The proposed method has shown its potential for determining uric acid concentration in biological samples. The proposed method provides a promising tool for clinical diagnose of uric acid and other fields of medical applications. It will of course be realized that whilst the above has been by way of an illustrative example of the invention, all such and other modifications and variations thereto, as would be apparent to persons skilled in the art, are deemed to fall within the broad scope and ambit of the inventions as claimed herein. Y2014/000256
1 1
REFERENCES
[1] CP. Huang, Y.K. Li, T.M. Chen, Biosens. Bioelectron. 22 (2007) 1835-1838. [2] J. Pan, S.S. Feng, Biomaterials 30 (2009)1 176-1183.
[3] M. Sun, L. Du, S. Gao, Y. Bao, S. Wang, Steroid 75 (2010) 400-403. [4] Y.Q. Wang, W.S. Zou, Talanta 85 (2011) 469-475.
[5] Z. Kaul, T. Yaguchi, S.C. Kaul, T. Hirano, R. Wadhwa, K. Taira, Cell Res. 13 (2003) 503-507.
[6] K.E. Sapsford, T. Pons, I.L. Medintz, H. Mattoussi, Sensors 6 (2006) 925-953. [7] W. Vastarella, R. Nicastri, Talanta 66 (2005) 627-633.
[8] D. Martinez-Perez, ML. Ferrer, C.R. Mate, Anal. Biochem. 322 (2003) 238-242. [9] F.F. Zhang, C.X. Li, X.H. Li, X.L. Wang, Q. Wan, Y.Z. Xian, L.T. Jin, K.
Yamamoto, Talanta 68 (2006) 1353-1358.
[10] C.R. Raj, T.J. Ohsaka, J. Electroanal. Chem. 540 (2003) 69-77. [11] R.C. Matos, L. Angnes, M.C.U. Ara'ujo, T.C.B. Saldanha, Analyst 125 (2000) 2011-2015.
[12] S.H. Huang, Y.C. Shih, C.Y. Wu, C.J. Yuan, Y.S. Yang, Y.K. Li, T.K. Wu, Biosens. Bioelectron. 19 (2004), 1627-1633. [13] M. Tabata, F. Chikaro, 0. Matashinge, M. Takashi, J. Appl. Biochem. 6 (1984) 251.
[14] J. Wang, T. Golden, P. Tuzhi, Anal. Chem. 59 (1987) 740-744.
[15] J. Galban, Y. Andreu, MJ. Almenara, S. de Marcos, J.R. Castillo, Talanta 54 (2001) 847-854.
[16] M. Koneswaran, R. Narayanaswamy, Sensors and Actuators B 139 (2009) 104- 109.
[17] M. Koneswaran, R. Narayanaswamy, Sensors and Actuators B 139 (2009) 91-96. [18] J. Tang, R.A. Marcus, The J. Chem. Physics 125 (2006) 044703-044708.
[19] X. Ji, J. Zheng, J. Xu, V.K. Rastogi, T.C. Cheng, J.J. DeFrank, R.M. Leblanc, The J. Physical Chem. B 109 (2005) 3793-3799.
[20] C. Dong, H. Qian, N. Fang, J. Ren, The J. Physical Chem. B 110 (2006) 11069- 11075.
[21] L.H. Cao, J. Ye, L.L. Tong, B. Tang, Chem. Eur. J. 14 (2008) 9633. [22] Y.C. Shiang, C.C. Huang, H.T. Chang, Chem. Commun. 14 (2009) 3437.
[23] F. Zhang, L. Chenxin, X. Li, Q. Wan, Y. Xian, L. Jin, K. Yamamoto, Analytica Chimica Acta 519 (2004) 155 - 160.
[24] M. Gao, S. Kirstein, H. Mohwald, A.L. Rogach, A. Komowski, A. Eychmuller, H.
Weller, The J. Physical Chem. B 102 (1998) 8360-8363. [25] D. Martinez-Perez, M.L. Ferrer, C.R. Mateo, Analytical Biochemistry 322 (2003), 238-242
[26] J.N. Miller and J.C. Miller, Statistics and Chemometrics for analytical chemistry, 4th Edition, Pearson Education Limited, Essex, England, 2000, pp. 42-51.

Claims

What is claimed is:
A hybrid system for the determination of uric acid comprising:
uricase and horseradish peroxidase enzyme ratio having specific reaction time; quantum dots as optical fluorescent indicator; and
a buffer solution for optimum pH value for hydrogen peroxide quenching effects of uric acid on the quantum dots fluorescence intensity.
The hybrid system for the determination of uric acid according to claim 1, wherein the uricase and horseradish peroxidase enzyme ratio is 5U:5U with a reaction time of 30 minutes.
The hybrid system for the determination of uric acid according to claim 1, wherein the quantum dots is mercaptopropionic acid (MPA)-capped cadmium sulphide (CdS).
The hybrid system for the determination of uric acid according to claim 3, wherein the mercaptopropionic acid (MPA)-capped cadmium sulphide (CdS) quantum dots have a fluorescence emission peak at wavelength of 565 nm.
The hybrid system for the determination of uric acid according to claim 1, wherein the optimum pH value for hydrogen peroxide quenching effects of uric acid on the quantum dots fluorescence intensity is pH 7. 6) The hybrid system for the determination of uric acid according to claim 1, wherein the buffer is phosphate solution.
7) The hybrid system for the determination of uric acid according to claim 1, wherein the quenching effects of hydrogen peroxide on the quantum dots is proportional to the concentrations of uric acid. 8) The hybrid system for the determination of uric acid according to claim 1, wherein the linear response of the hybrid system was obtained at uric acid concentration range of 125-1000 μΜ with detection limit of 125 μΜ.
PCT/MY2014/000256 2013-08-27 2014-10-23 Application of enzyme-quantum dots hybrid system for the determination of uric acid WO2015047079A2 (en)

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EP4053951A1 (en) 2021-02-25 2022-09-07 Aarhus Universitet System and method for balancing a vanadium redox flow battery
CN115612486A (en) * 2022-10-31 2023-01-17 扬州大学 Cobalt/manganese dioxide quantum dot with uricase activity and preparation method thereof

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CN105784660A (en) * 2016-04-05 2016-07-20 广西师范学院 Method for detecting concentration of horseradish peroxidase by utilizing water-soluble InP/ZnS QDs probe
CN106872422A (en) * 2016-12-30 2017-06-20 锦州医科大学 The method of uric acid in quantum dots characterization body fluid
CN110726707A (en) * 2019-10-30 2020-01-24 南京医科大学 Based on N-Ti3C2Composite nano probe of QDs and o-phenylenediamine oxide and ratiometric fluorescence detection method thereof
CN110726707B (en) * 2019-10-30 2022-08-23 南京医科大学 Based on N-Ti 3 C 2 Composite nano probe of QDs and o-phenylenediamine oxide and detection method thereof
EP4053951A1 (en) 2021-02-25 2022-09-07 Aarhus Universitet System and method for balancing a vanadium redox flow battery
CN115612486A (en) * 2022-10-31 2023-01-17 扬州大学 Cobalt/manganese dioxide quantum dot with uricase activity and preparation method thereof
CN115612486B (en) * 2022-10-31 2024-02-13 扬州大学 Cobalt/manganese dioxide quantum dot with uricase-like activity and preparation method thereof

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