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Dopamine functionalized CuInS2 quantum dots as a fluorescence probe for urea
Sensors and Actuators B 191 (2014) 246–251
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Dopamine functionalized CuInS2 quantum dots as a fluorescence probe for urea Siyu Liu, Fanping Shi, Lu Chen, Xingguang Su ∗ Department of Analytical Chemistry, College of Chemistry, Jilin University, Changchun 130012, China
a r t i c l e
i n f o
Article history: Received 27 June 2013 Received in revised form 10 September 2013 Accepted 11 September 2013 Available online 25 September 2013 Keywords: CuInS2 quantum dots Dopamine Fluorescence quenching Urea Urase
a b s t r a c t In this paper, a simple, convenient and selective urea-biosensing system with dopamine-functionalized CuInS2 quantum dots (QDs) as the fluorescence probe was developed. Water-soluble CuInS2 QDs capped by 3-mercaptopropionic acid (MPA) was directly synthesized in aqueous solution, and then it was linked to dopamine to form the dopamine-functionalized CuInS2 QDs (DA-CuInS2 QDs) fluorescence probe. When the pH value of DA-CuInS2 QDs solution was adjusted from neutral to alkalinity, the dopamine on the surface of QDs would change into quinone, which would lead to the fluorescence quenching of DA-CuInS2 QDs due to the charge transfer interactions between QDs and the proximal quinone. Based on the fact that the hydrolysis of urea in the presence of urase would release OH− to change the pH value of the solution, so the fluorecence intensity of DA-CuInS2 QDs could be linked to the enzymatic degradation of urea. The novel urea-biosensing system could effectively probe urea in the dynamic concentration range from 0.2 to 6 mmol/L. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Urea, as a fertilizer, and animal feeding stuff additive, is massproduced every year and widely applied in agriculture and chemical industry [1,2]. And not only that, urea is the major excretory product of protein metabolism and non-protein nitrogenous substance in body [3]. Urea in blood or in urine is an important marker in the diagnosis of renal and liver diseases [4]. It is necessary to perform the analysis of urea content in serum or urine before diagnosing the different dysfunctions of the patients. The urea concentration in serum of healthy adults may be in the range from 30 to 80 mmol/L [5]. Due to the important effects of urea on human health, to develop a sensitive and fast method for the determination of urea has attracted more attention. So far, urea can be measured by several techniques, such as, conductance [6,7], potentiometry [8,9], amperometry [10,11], and spectrophotometry [1,12]. However, complicated operation, long operation time and high cost were found in these methods. In recent years, a fluorescence measurement has been successfully applied in the determination of some small biological molecules owing to its operational simplicity, high sensitivity and real-time monitoring [13–24]. So far, there are only a few reports on the fluorescence probe for the determination of urea. Huang et al. proposed an assay system with
∗ Corresponding author. Tel.: +86 431 85168352. E-mail address: [email protected] (X. Su). 0925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2013.09.056
urase as a catalyst and CdSe/ZnS QDs capped by mercaptosuccinic acid (MSA) as an indicator for quantitative analysis of urea, and the fluorescence intensity of CdSe/ZnS QDs could be enhanced in alkaline environment after the enzyme–urea reaction [25]. Ruedas-Rama developed an enzyme-linked nanosphere sensor to probe urea based on fluorescence of QDs responds to the pH value changes after the enzyme–urea reaction [4]. As well-known, the urase-catalyzed hydrolysis of urea releases NH4 + , OH− and HCO3 − ions as products, and the pH value of the system gradually increase with the urea hydrolysis. Therefore, a high sensitive pH sensor is the key to develop the fluorescence measurements for urea. As previous reports, QDs capped with some kinds of mercaptoalkanoic acids provided an obvious route to obtain pH sensor [25–29], and some other alternative ligands were introduced to control pH sensitivity of QDs [28,29]. Liu and coworkers reported the fluorescence of a mercaptoacetic acid-capped CdSe/ZnSe/ZnS QDs solution increased by around fivefold when the pH changed from 4 to 10 [27]. Tomasulo et al. developed a strategy to switch the fluorescence of CdSe/ZnS core–shell QDs with pH changes of systems that is based on the photoinduced transfer of either energy from CdSe/ZnS QDs to [1,3]oxazine ligands or electrons from the organic to the inorganic components [28]. Dopamine (DA), as one of the most significant catecholamine neurotransmitters, influences a wide variety of human’s motivated behaviors, attention span, and neuronal plasticity [30–33]. Dopamine and some of its derivatives exhibit complex redox properties with pH-tunable oxidation and reduction potentials [34–36]. Some researchers have reported that the oxidation product
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benzoquinone of dopamine could effectively quench the fluorescence of QDs, and such fluorescence quenching process could be attributed to charge transfer between the QDs and proximal benzoquinone [34,36–39]. However, the traditional cadmium-based QDs are toxic for biological systems and eventually will cause serious environmental problems due to the leak of cadmium. Recently, a novel kind of I-III-VI CuInS2 QDs that does not contain any toxic Class A element (Cd, Pb, and Hg) or B element (Se and As) has attracted considerable interests [40–43]. In previous report, we presented the one-pot synthesis of water-soluble CuInS2 QDs capped with 3-mercaptopropionic acid (MPA) [44]. In this paper, MPA capped CuInS2 QDs are covalently bonded to dopamine via 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) as coupling reagents. The fluorescence of the obtained dopamine functionalized CuInS2 QDs (DA-CuInS2 QDs) dramatically reduced with the increased pH value of the environments. Based on the fact that the urease-catalyzed hydrolysis of urea could release OH− to increase the pH value of the environments, we developed a simple, convenient and highly sensitive fluorescence method for the detection of urea utilizing the pH sensitive dopamine functionalized CuInS2 QDs as the fluorescence probe.
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2.4. Preparation of dopamine functionalized CuInS2 QDs The conjugation of dopamine to CuInS2 QDs was performed utilizing EDC and NHS as coupling reagents according to previous reports [39,45]. Briefly, the CuInS2 QDs solution was mixed with EDC and NHS at room temperature, and then dopamine was added into the solution, stirring for more than 3 h (the mole ratio of QD to NHS, EDC, dopamine was 1:1500:1500:1000). The DA-CuInS2 QDs were purified by precipitation with addition of THF to the reaction system, and the purified DA-CuInS2 QDs powder were dissolved in aqueous solution, and stored in the darkroom. 2.5. Detection of urea by DA-CuInS2 QDs For urea determination, 200 L urase solution (12.5 mg/mL, dissolved in 10 mmol/L PBS pH 7.0) and different amount of urea were added in to a 2 mL calibrated test tube, shaken thoroughly for 1 min. And then 25 L DA-CuInS2 QDs solution (0.12 mg/mL) was added into the test tube, diluted to the mark with deionized water, shaken thoroughly and equilibrated for 30 min. The fluorescence spectra of the mixture were recorded from 580 nm to 850 nm, and the sampling interval was 1.0 nm. The slit widths of excitation and emission were both 10 nm. The fluorescence (FL) intensity of the maximum emission peak was used for the quantitative analysis of the urea.
2. Experiment 2.6. Preparation of serum samples 2.1. Apparatus The fluorescence spectra were obtained by using a Shimadzu RF-5301 PC spectrofluorophotometer equipped with a xenon lamp using right-angle geometry. UV–vis absorption spectra were obtained by a Varian GBC Cintra 10e UV-vis spectrometer. In both experiments, a 1 cm path-length quartz cuvette was used. 2.2. Reagents All reagents were of at least analytical grade. The water used in all experiments had a resistivity higher than 18 M cm−1 . Copper (II) chloride dihydrate (CuCl2 ·2H2 O), sodium hydroxide (NaOH), sulfourea (CS(NH2 )2 ), urea, tetrahydrofuran (THF), sodium dehydrogenized phosphate (NaH2 PO4 ) and disodium hydrogen phosphate (Na2 HPO4 ) were purchased from Shanghai Qingxi Technology Co., Ltd. Dopamine, indium (III) chloride tetrahydrate (InCl3 ·4H2 O), MPA, EDC, NHS, and urease (1 U/mg) were purchased from Sigma–Aldrich Corporation. 2.3. Preparation of MPA-capped CuInS2 QDs MPA-capped CuInS2 QDs were synthesized in aqueous solution, according to our previous report [44]. 0.15 mmol CuCl2 ·2H2 O and InCl3 ·4H2 O were dissolved in 10.5 mL distilled water, and then 1.8 mmol MPA was injected into the solution. The pH value of the mixture solution was adjusted to 11.3 by adding 2 mol/L NaOH solution with stirring during this process. After stirring for 10 min, 0.30 mmol CS(NH2 )2 was dissolved in above solution. The previous process was all finished at room temperature, and then the mixture solution was finally transferred into a 15 mL autoclave. Then it was heated and maintained at 150 ◦ C for 21 h and then cooled down to room temperature by a natural process. Enthanol was added to the stock solution to obtain CuInS2 QDs precipitate, and the process was repeated three times. The unreacted residues were removed by the cycled washing. The purified CuInS2 QDs were dissolved in PBS (2 mmol/L, pH 7.0), and stored in the darkroom. The final CuInS2 QDs solution concentration was 0.15 mmol/L.
Drug-free human blood samples were collected from healthy volunteers and then centrifuged at 10 000 rpm for 10 min after standing for 2 h at room temperature. 1 mL obtained serum samples and 2 mg urase were mixed and incubated for 24 h at 35 ◦ C to hydrolyze the inherent urea in the serum. And then 3 mL 15% trichloroacetic acid was added into the serum to destroy the activity of urase, which would irreparably lose its catalytic activity under strong acid condition, and precipitate proteins of the serum [46,47]. After vigorously shaking for 15 min, the mixture was centrifuged at 10 000 rpm for 10 min at 4 ◦ C. The obtained supernatant was adjusted to pH 7.0 using NaOH solution and then diluted by 50 times with deionized water. Different concentrations of urea were added to the diluted serum samples to prepare the spiked samples. 3. Results and discussions 3.1. DA-CuInS2 QDs fluorescence probe As well-known that, the fluorescence properties of QDs had a close connection to their surface capped layers [13,48]. As shown in Scheme 1, in this study, we used dopamine to modify MPA capped CuInS2 QDs to form the dopamine-functionalized CuInS2 QDs fluorescence probe. The urea would release OH− with urase as catalysts that could induce changes from dopamine to its corresponding quinone. And the energy transfer between the QDs and proximal quinone would lead to the fluorescence quenching of DA-CuInS2 QDs, so the DA-CuInS2 QDs fluorescence probe could be utilized for the determination of urea. Fig. 1 shows the UV–vis absorption and fluorescence emission spectra of MPA-capped CuInS2 QDs and DA-CuInS2 QDs respectively. It could be seen that after conjugation with dopamine, the fluorescence emission peak exhibited a blue-shift from 660 nm to 630 nm, which was attributed to that the surface capping layer of the CuInS2 QDs was changed from the negatively charged acetate to the electron donor dopamine [39,45]. Fig. S1A shows the FT-IR spectra of the MPA-capped CuInS2 QDs (curve a) and DA-CuInS2 QDs (curve b). As curve a in Fig. S1A shown, the majority of MPA functional groups could be clearly found through the C O stretching peak (1730 cm−1 ), and C H
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Scheme 1. The schematic illustration of the synthetic process of DA-CuInS2 QDs and the detection of urea.
stretching mode (2850 cm−1 , 2920 cm−1 , 2960 cm−1 ). The characteristic peak of S H did not appear within 2550–2680 cm−1 , which might be caused by the covalent bonds between thiols and metal. As curve b in Fig. S1A shown, the amide I band (1660 cm−1 ), amide II (1540 cm−1 ) and amide III (1400 cm−1 ) band for CONH group appeared, which was due to the conjugation of DA to the MPA capped CuInS2 QDs. UV–vis absorption spectra (230–400 nm) of the DA-CuInS2 QDs were performed. As shown in Fig. S1B, the typical UV–vis absorption peak around 280 nm belonging to DA was observed in the DA-CuInS2 QDs, and not observed in the original MPA-capped CuInS2 QDs. It was further confirmed the presence of dopamine after the conjugation,
other possible interference on the fluorescence of DA-CuInS2 QDs, we respectively investigated the effect of urea, NH4 + , HCO3 − and urase on the fluorescence of DA-CuInS2 QDs. Fig. S3 shows the fluorescence intensity of DA-CuInS2 QDs with different concentration of urea, NH4 + , HCO3 − (in the range from 0 to 10 mmol/L) or urase (in the range from 0 to 1.25 mg/mL). It can be seen that urea, only NH4 + , HCO3 − or urase itself cannot induce obviously fluorescence quenching of the DA-CuInS2 QDs. So it was further confirmed that we could detect urea based on the fluorescence quenching of DACuInS2 QDs induced by OH− that was released from the hydrolysis of urea in the presence of urase. 3.3. Optimization for urea detection
3.2. The influence of pH on the fluorescence of DA-CuInS2 QDs The charge transfer interactions between fluorescent QDs and redox active dopamine have been extensively investigated [34,36]. In this work, we studied the effect of pH on the fluorescence properties of DA-CuInS2 QDs. As shown in Fig. 2, it could be seen that the fluorescence intensity of DA-CuInS2 QDs decreased with the increasing of the pH from 7.0 to 9.93, and when the pH increased to be 9.93, the fluorescence was almost entirely quenched. The significant fluorescence quenching was due to that the oxygen-catalytic oxidation of dopamine potentials was gradually reduced as the increased pH value from 7.0 to 9.93, and the equilibrium favored the transformation of dopamine to its oxidation product quinone at high pH. And the transfer of electrons from a photoexcited QDs to the lowest unoccupied molecular orbital of the proximal quinone acceptor would result in the fluorescence quenching of DA-CuInS2 QDs [34,36]. As shown in Fig. S2, under the acid conditions, the fluorescence intensity of DA-CuInS2 QDs did not exhibit significant variation with the decreasing pH value of the system. As well-known, the urea could release NH4 + , OH− and HCO3 − ions as products with urase as the catalyst. In order to exclude the
Fig. 1. The UV–vis absorption and fluorescence emission spectra of MPA-capped CuInS2 QDs (dash line) and DA-CuInS2 QDs (solid line).
As described in Eq. (1), the urea could release ionic product NH4 + , HCO3 − and OH− in the presence of urase. CO(NH2 )2 + 3H2 O = 2NH4 + + HCO3 − + OH−
(1)
As shown in Fig. 2, the fluorescence of DA-CuInS2 QDs would be quenched when pH of the system changed from neutral to alkalinity. Therefore, DA-CuInS2 QDs could be utilized as a fluorescence probe for the determination of urea with urase as catalyst. As wellknown that the urase plays a key role in the hydrolysis of urea. Therefore, choosing the appropriate concentration of urase could improve the sensitivity for urea detection by DA-CuInS2 QDs probe. In this work, the fluorescence intensity ratio F/F0 (F and F0 are the fluorescence intensity of DA-CuInS2 QDs with or without urea) of DA-CuInS2 QDs around 630 nm quenched by 2 mmol/L urea in the presence of different urase concentration (0.08–1.25 mg/mL) were studied, and the results were shown in Fig. 3. It can be seen that
Fig. 2. The fluorescence (FL) spectra of DA-CuInS2 QDs after the addition of different amounts of NaOH with the pH value of the system changed from 7.00 to 9.93 (pH = 7.00, 7.16, 7.27, 7.58, 7.96, 8.55, 8.95, 9.31, 9.70, 9.93). Inset: the plot of fluorescence quenching ratios of F/F0 at 630 nm versus the pH value of the system at room temperature.
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Fig. 3. The relationship between the fluorescence intensity ratio F/F0 of DA-CuInS2 QDs and the reaction time. F and F0 are the fluorescence intensity of DA-CuInS2 QDs with or without urea. Conditions: 2 mmol/L urea and various urase concentrations (0.08, 0.21, 0.42, 0.63, 1.25 mg/mL).
the fluorescence intensity ratio F/F0 of DA-CuInS2 QDs decreased slightly with the increasing of reaction time in the presence of low concentration of urase (0.08 or 0.21 mg/mL). When the urase concentration increased in the assay system (0.42–1.25 mg/mL), more dramatic changes of fluorescence intensity ratios F/F0 were observed. The gradually decreased fluorescence intensity ratios F/F0 with the increasing of reaction time clearly suggests that the urea was gradually decomposed to release OH− by catalysis of urase, and the increased concentrations of OH− in the assay system would result in the more obvious fluorescence quenching of DA-CuInS2 QDs. As Fig. 3 showed when the urase cencentration increased from 0.63 mg/mL to 1.25 mg/mL, the fluorescence intensity ratios (F/F0 ) after 30 min of enzyme hydrolysis almost reached the same. The results indicated that the urase concentration of 1.25 mg/mL was sufficient in this urea molecules hydrolysis system, and it was corresponding with previous reports [4,25]. In this paper, we investigated systematically the effect of the reaction temperature on this urea detection system, and the results were shown in Fig. S4. As curve a in Fig. S2 shown, the fluoresce intensity of DA-CuInS2 QDs decreased gradually with the increase of reaction temperature from 15 ◦ C to 40 ◦ C, which was due to that the increase of reaction temperature would increase the rate of oxidation and reduce the dopamine potential for oxidation and shifted the equilibrium toward the production of quinones [36,49–51]. Urase is an enzyme in organisms, and its activities increased gradually with the increase of temperature at a given range that was corresponding with fluorescence intensity changes of DA-CuInS2 QDs–urea–urase system (curve b). Considering the stability of DACuInS2 QDs, the activities of urase and being simple to operate, room temperature 20 ◦ C was chosen as the reaction temperature in our urea detection system. 3.4. Fluorescence detection of urea using the DA-CuInS2 QDs As shown in Fig. 4, the fluorescence of DA-CuInS2 QDs decreased as the urea concentration increased from 5 mol/L to 12 mmol/L in the presence of 1.25 mg/mL urase as catalyst. Fig. 4 inset describes the relationship between the fluorescence intensity ratio F/F0 (F and F0 are the fluorescence intensity of DA-CuInS2 QDs with or without urea) and the logarithm of urea concentration (0.005–12 mmol/L, logarithm value −2.30 to 1.08). It could be seen that when the concentration of urea was 12 mmol/L, the fluorescence intensity of DA-CuInS2 QDs would decrease to about 20% of original
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Fig. 4. The fluorescence (FL) spectra of DA-CuInS2 QDs upon the addition of different concentration of urea in the range of 0–12 mmol/L (0, 0.005, 0.020, 0.050, 0.10, 0.20, 0.50, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0, 12.0 mmol/L) in the presence of 1.25 mg/mL urase. Inset: the plot of fluorescence quenching ratios of F/F0 at 630 nm versus the concentration of urea at room temperature.
fluorescence intensity. The obviously fluorescence quenching induced by urea with urase as catalysts was due to the hydrolysis of urea, which released OH− to change the pH value of the system, and it favored easier transformation of dopamine to its oxidation product quinone. And then the transfer of electrons from QDs to the proximal quinone acceptor would result in the fluorescence quenching of DA-CuInS2 QDs. The proposed urea-biosensing system could effectively probe urea in the dynamic range from 0.2 to 6 mmol/L. As shown in Table 1, we compared the detecting limits and dynamic range of urea with various methodologies including potentiometry [10,52], amperometry [8], fluorescence measurements [4,25] and our proposed detection system. It can be seen that the proposed method offered a similar detecting limit and wide dynamic range for the detection of urea. Compared with previous reports about urea sensors, the DA-CuInS2 QD-based biosensor offered many advantages such as simple operations, low cost, without enzyme immobilization, good selectivity and sensitivity. 3.5. Effect of foreign substance In this paper, we studied the effect of some physiological ions and molecules on our proposed urea fluorescence detection system. It could be found that from Fig. S5A, the fluorescence intensity of DA-CuInS2 QDs with 1.25 mg/mL urase added by 2 mmol/L foreign substance K+ , Na+ , Ca2+ , Mg2+ , glucose (Glu), glucine (Gly), threonine (Thr) or lysine (lys) remained nearly constant, and only after the addition of urea, the fluorescence intensity showed obvious decreasing. The results indicated that the proposed urea detection system showed the high selectivity to urea. In Fig. S5B, we further investigated the fluorescence response of DA-CuInS2 QDs to 2 mmol/L urea with 1.25 mg/L urase in the presence of 2 mmol/L foreign substance K+ , Na+ , Ca2+ , Mg2+ , Glu, Gly, Thr or lys. It could be seen from Fig. S5B that even in the presence of 2 mmol/L interference, the DA-CuInS2 QDs sensor still work the same. The results showed that the proposed urea detection system could provide well ability of resisting interference from physiological ions and molecules. 3.6. Detection of urea in real samples In order to test the applicability of the proposed method, it was applied to determinate urea in human serum samples which spiked with different concentration of urea. The results obtained by
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Table 1 Comparison of performance of different urea sensors. Type
Sensing system
Detection limit (mmol/L)
Reference
Potentiometry
Poly(N-vinylcarbazole)/stearic acid Langmuir–Blodgett films Mercaptohydroquinone-modified gold electrode
10–68 0.2–5
5 0.2
[52] [10]
Amperometry
Urease + polyurethane–acrylate polytoluidine blue film
0.2–0.8
0.02
[8]
Fluorescence
Urea/MSA-CdSe/ZnS QDs Mercaptopropionic capped QDs–PAH-CaR Dopamine functionalized CuInS2 QDs
0.01–120 0.4–25 0.2–6
0.01 0.4 0.1
[25] [4] This work
standard addition method were shown in Table S1. The accuracy of the proposed method was evaluated by determining the recoveries of urea in real samples. It could be found that the average recoveries of urea in the real samples was in the range of 98–102% and the RSD was lower than 3.2%. The above results demonstrated that the potential applicability of the DA-CuInS2 QD-based fluorescence probe for the detection of urea in human serum samples. 4. Conclusion We have demonstrated the dopamine functionalized CuInS2 QDs that was sensitive to the pH values changes can be utilized as an effective fluorescence probe for the determination of urea. The fluorescence of the DA-CuInS2 QDs was quenched by urea in the range from 0.2 to 6 mmol/L with urase as the catalyst. The proposed method was successfully applied to the detection of urea in human serum sample with satisfactory results. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (Nos. 20875036 and 21075050) and the Science and Technology Development Project of Jilin Province, China (No. 20110334). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2013.09.056. References [1] B. Kovács, G. Nagy, R. Dombi, K. Tóth, Optical biosensor for urea with improved response time, Biosensors and Bioelectronics 18 (2003) 111–118. [2] J.M. Garrido, R. Mendez, J.M. Lema, Treatment of wastewaters from a formaldehyde-urea adhesives factory, Water Science and Technology 42 (2000) 293–300. [3] R. Doong, H.M. Shih, Array-based titanium dioxide biosensors for ratiometric determination of glucose, glutamate and urea, Biosensors and Bioelectronics 25 (2010) 1439–1446. [4] M.J. Ruedas-Rama, E.A.H. Hall, Analytical nanosphere sensors using quantum dot–enzyme conjugates for urea and creatinine, Analytical Chemistry 82 (2010) 9043–9049. [5] J.C. Chen, J.C. Chou, T.P. Sun, S.K. Hsiung, Portable urea biosensor based on the extended-gate field effect transistor, Sensors and Actuators: B Chemical 91 (2003) 180–186. [6] P.S. Chaudhari, A. Gokarna, M. Kulkarni, M.S. Karve, S.V. Bhoraskar, Porous silicon as an entrapping matrix for the immobilization of urease, Sensors and Actuators: B Chemical 107 (2005) 258–263. [7] W.Y. Lee, S.R. Kim, T.H. Kim, K.S. Lee, M.C. Shin, J.K. Park, Sol–gel-derived thickfilm conductometric biosensor for urea determination in serum, Analytica Chimica Acta 404 (2000) 195–203. [8] I. Vostiar, J. Tkac, E. Sturdik, P. Gemeiner, Amperometric urea biosensor based on urease and electropolymerized toluidine blue dye as a pH-sensitive redox probe, Bioelectrochemistry 56 (2002) 113–115. [9] A.P. Soldatkin, J. Montoriol, W. Sant, C. Martelet, N. Jaffrezic-Renault, A novel urea sensitive biosensor with extended dynamic range based on recombinant urease and ISFETs, Biosensors and Bioelectronics 19 (2003) 131–135. [10] F. Mizutani, S. Yabuki, Y. Sato, Voltammetric enzyme sensor for urea using mercaptohydroquinone-modified gold electrode as the base transducer, Biosensors and Bioelectronics 12 (1997) 321–328.
Dynamic range (mmol/L)
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Biographies Siyu Liu is currently carrying out his graduate work for his doctor degree under the guidance of Professor Su in Jilin University. His research focuses on the synthesis and functionalization of quantum dots and their application on sensors for metal ion and small molecules. Fanping Shi is currently carrying out her graduate work for her masters degree under the guidance of Professor Su in Jilin University. Her research focuses on the synthesis and functionalization of quantum dots and their application on sensors for metal ion and small molecules. Lu Chen received her bachelor degree in Institute of Chemistry Jilin University. Her research focuses on the application of quantum dots on sensors for small molecules. Xingguang Su is a professor at the Department of Analytical Chemistry at the College of Chemistry, Jilin University. She received her master degree from Jilin University (China) in 1992 and her doctor degree from Jilin University (China) in 1999. Her research focuses on the synthesis, characterization, functionalization and application of quantum dots and quantum dots-tagged microspheres in biomedicine.
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Dopamine functionalized CuInS2 quantum dots as a fluorescence probe for urea Siyu Liu, Fanping Shi, Lu Chen, Xingguang Su ∗ Department of Analytical Chemistry, College of Chemistry, Jilin University, Changchun 130012, China
a r t i c l e
i n f o
Article history: Received 27 June 2013 Received in revised form 10 September 2013 Accepted 11 September 2013 Available online 25 September 2013 Keywords: CuInS2 quantum dots Dopamine Fluorescence quenching Urea Urase
a b s t r a c t In this paper, a simple, convenient and selective urea-biosensing system with dopamine-functionalized CuInS2 quantum dots (QDs) as the fluorescence probe was developed. Water-soluble CuInS2 QDs capped by 3-mercaptopropionic acid (MPA) was directly synthesized in aqueous solution, and then it was linked to dopamine to form the dopamine-functionalized CuInS2 QDs (DA-CuInS2 QDs) fluorescence probe. When the pH value of DA-CuInS2 QDs solution was adjusted from neutral to alkalinity, the dopamine on the surface of QDs would change into quinone, which would lead to the fluorescence quenching of DA-CuInS2 QDs due to the charge transfer interactions between QDs and the proximal quinone. Based on the fact that the hydrolysis of urea in the presence of urase would release OH− to change the pH value of the solution, so the fluorecence intensity of DA-CuInS2 QDs could be linked to the enzymatic degradation of urea. The novel urea-biosensing system could effectively probe urea in the dynamic concentration range from 0.2 to 6 mmol/L. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Urea, as a fertilizer, and animal feeding stuff additive, is massproduced every year and widely applied in agriculture and chemical industry [1,2]. And not only that, urea is the major excretory product of protein metabolism and non-protein nitrogenous substance in body [3]. Urea in blood or in urine is an important marker in the diagnosis of renal and liver diseases [4]. It is necessary to perform the analysis of urea content in serum or urine before diagnosing the different dysfunctions of the patients. The urea concentration in serum of healthy adults may be in the range from 30 to 80 mmol/L [5]. Due to the important effects of urea on human health, to develop a sensitive and fast method for the determination of urea has attracted more attention. So far, urea can be measured by several techniques, such as, conductance [6,7], potentiometry [8,9], amperometry [10,11], and spectrophotometry [1,12]. However, complicated operation, long operation time and high cost were found in these methods. In recent years, a fluorescence measurement has been successfully applied in the determination of some small biological molecules owing to its operational simplicity, high sensitivity and real-time monitoring [13–24]. So far, there are only a few reports on the fluorescence probe for the determination of urea. Huang et al. proposed an assay system with
∗ Corresponding author. Tel.: +86 431 85168352. E-mail address: [email protected] (X. Su). 0925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2013.09.056
urase as a catalyst and CdSe/ZnS QDs capped by mercaptosuccinic acid (MSA) as an indicator for quantitative analysis of urea, and the fluorescence intensity of CdSe/ZnS QDs could be enhanced in alkaline environment after the enzyme–urea reaction [25]. Ruedas-Rama developed an enzyme-linked nanosphere sensor to probe urea based on fluorescence of QDs responds to the pH value changes after the enzyme–urea reaction [4]. As well-known, the urase-catalyzed hydrolysis of urea releases NH4 + , OH− and HCO3 − ions as products, and the pH value of the system gradually increase with the urea hydrolysis. Therefore, a high sensitive pH sensor is the key to develop the fluorescence measurements for urea. As previous reports, QDs capped with some kinds of mercaptoalkanoic acids provided an obvious route to obtain pH sensor [25–29], and some other alternative ligands were introduced to control pH sensitivity of QDs [28,29]. Liu and coworkers reported the fluorescence of a mercaptoacetic acid-capped CdSe/ZnSe/ZnS QDs solution increased by around fivefold when the pH changed from 4 to 10 [27]. Tomasulo et al. developed a strategy to switch the fluorescence of CdSe/ZnS core–shell QDs with pH changes of systems that is based on the photoinduced transfer of either energy from CdSe/ZnS QDs to [1,3]oxazine ligands or electrons from the organic to the inorganic components [28]. Dopamine (DA), as one of the most significant catecholamine neurotransmitters, influences a wide variety of human’s motivated behaviors, attention span, and neuronal plasticity [30–33]. Dopamine and some of its derivatives exhibit complex redox properties with pH-tunable oxidation and reduction potentials [34–36]. Some researchers have reported that the oxidation product
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benzoquinone of dopamine could effectively quench the fluorescence of QDs, and such fluorescence quenching process could be attributed to charge transfer between the QDs and proximal benzoquinone [34,36–39]. However, the traditional cadmium-based QDs are toxic for biological systems and eventually will cause serious environmental problems due to the leak of cadmium. Recently, a novel kind of I-III-VI CuInS2 QDs that does not contain any toxic Class A element (Cd, Pb, and Hg) or B element (Se and As) has attracted considerable interests [40–43]. In previous report, we presented the one-pot synthesis of water-soluble CuInS2 QDs capped with 3-mercaptopropionic acid (MPA) [44]. In this paper, MPA capped CuInS2 QDs are covalently bonded to dopamine via 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) as coupling reagents. The fluorescence of the obtained dopamine functionalized CuInS2 QDs (DA-CuInS2 QDs) dramatically reduced with the increased pH value of the environments. Based on the fact that the urease-catalyzed hydrolysis of urea could release OH− to increase the pH value of the environments, we developed a simple, convenient and highly sensitive fluorescence method for the detection of urea utilizing the pH sensitive dopamine functionalized CuInS2 QDs as the fluorescence probe.
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2.4. Preparation of dopamine functionalized CuInS2 QDs The conjugation of dopamine to CuInS2 QDs was performed utilizing EDC and NHS as coupling reagents according to previous reports [39,45]. Briefly, the CuInS2 QDs solution was mixed with EDC and NHS at room temperature, and then dopamine was added into the solution, stirring for more than 3 h (the mole ratio of QD to NHS, EDC, dopamine was 1:1500:1500:1000). The DA-CuInS2 QDs were purified by precipitation with addition of THF to the reaction system, and the purified DA-CuInS2 QDs powder were dissolved in aqueous solution, and stored in the darkroom. 2.5. Detection of urea by DA-CuInS2 QDs For urea determination, 200 L urase solution (12.5 mg/mL, dissolved in 10 mmol/L PBS pH 7.0) and different amount of urea were added in to a 2 mL calibrated test tube, shaken thoroughly for 1 min. And then 25 L DA-CuInS2 QDs solution (0.12 mg/mL) was added into the test tube, diluted to the mark with deionized water, shaken thoroughly and equilibrated for 30 min. The fluorescence spectra of the mixture were recorded from 580 nm to 850 nm, and the sampling interval was 1.0 nm. The slit widths of excitation and emission were both 10 nm. The fluorescence (FL) intensity of the maximum emission peak was used for the quantitative analysis of the urea.
2. Experiment 2.6. Preparation of serum samples 2.1. Apparatus The fluorescence spectra were obtained by using a Shimadzu RF-5301 PC spectrofluorophotometer equipped with a xenon lamp using right-angle geometry. UV–vis absorption spectra were obtained by a Varian GBC Cintra 10e UV-vis spectrometer. In both experiments, a 1 cm path-length quartz cuvette was used. 2.2. Reagents All reagents were of at least analytical grade. The water used in all experiments had a resistivity higher than 18 M cm−1 . Copper (II) chloride dihydrate (CuCl2 ·2H2 O), sodium hydroxide (NaOH), sulfourea (CS(NH2 )2 ), urea, tetrahydrofuran (THF), sodium dehydrogenized phosphate (NaH2 PO4 ) and disodium hydrogen phosphate (Na2 HPO4 ) were purchased from Shanghai Qingxi Technology Co., Ltd. Dopamine, indium (III) chloride tetrahydrate (InCl3 ·4H2 O), MPA, EDC, NHS, and urease (1 U/mg) were purchased from Sigma–Aldrich Corporation. 2.3. Preparation of MPA-capped CuInS2 QDs MPA-capped CuInS2 QDs were synthesized in aqueous solution, according to our previous report [44]. 0.15 mmol CuCl2 ·2H2 O and InCl3 ·4H2 O were dissolved in 10.5 mL distilled water, and then 1.8 mmol MPA was injected into the solution. The pH value of the mixture solution was adjusted to 11.3 by adding 2 mol/L NaOH solution with stirring during this process. After stirring for 10 min, 0.30 mmol CS(NH2 )2 was dissolved in above solution. The previous process was all finished at room temperature, and then the mixture solution was finally transferred into a 15 mL autoclave. Then it was heated and maintained at 150 ◦ C for 21 h and then cooled down to room temperature by a natural process. Enthanol was added to the stock solution to obtain CuInS2 QDs precipitate, and the process was repeated three times. The unreacted residues were removed by the cycled washing. The purified CuInS2 QDs were dissolved in PBS (2 mmol/L, pH 7.0), and stored in the darkroom. The final CuInS2 QDs solution concentration was 0.15 mmol/L.
Drug-free human blood samples were collected from healthy volunteers and then centrifuged at 10 000 rpm for 10 min after standing for 2 h at room temperature. 1 mL obtained serum samples and 2 mg urase were mixed and incubated for 24 h at 35 ◦ C to hydrolyze the inherent urea in the serum. And then 3 mL 15% trichloroacetic acid was added into the serum to destroy the activity of urase, which would irreparably lose its catalytic activity under strong acid condition, and precipitate proteins of the serum [46,47]. After vigorously shaking for 15 min, the mixture was centrifuged at 10 000 rpm for 10 min at 4 ◦ C. The obtained supernatant was adjusted to pH 7.0 using NaOH solution and then diluted by 50 times with deionized water. Different concentrations of urea were added to the diluted serum samples to prepare the spiked samples. 3. Results and discussions 3.1. DA-CuInS2 QDs fluorescence probe As well-known that, the fluorescence properties of QDs had a close connection to their surface capped layers [13,48]. As shown in Scheme 1, in this study, we used dopamine to modify MPA capped CuInS2 QDs to form the dopamine-functionalized CuInS2 QDs fluorescence probe. The urea would release OH− with urase as catalysts that could induce changes from dopamine to its corresponding quinone. And the energy transfer between the QDs and proximal quinone would lead to the fluorescence quenching of DA-CuInS2 QDs, so the DA-CuInS2 QDs fluorescence probe could be utilized for the determination of urea. Fig. 1 shows the UV–vis absorption and fluorescence emission spectra of MPA-capped CuInS2 QDs and DA-CuInS2 QDs respectively. It could be seen that after conjugation with dopamine, the fluorescence emission peak exhibited a blue-shift from 660 nm to 630 nm, which was attributed to that the surface capping layer of the CuInS2 QDs was changed from the negatively charged acetate to the electron donor dopamine [39,45]. Fig. S1A shows the FT-IR spectra of the MPA-capped CuInS2 QDs (curve a) and DA-CuInS2 QDs (curve b). As curve a in Fig. S1A shown, the majority of MPA functional groups could be clearly found through the C O stretching peak (1730 cm−1 ), and C H
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Scheme 1. The schematic illustration of the synthetic process of DA-CuInS2 QDs and the detection of urea.
stretching mode (2850 cm−1 , 2920 cm−1 , 2960 cm−1 ). The characteristic peak of S H did not appear within 2550–2680 cm−1 , which might be caused by the covalent bonds between thiols and metal. As curve b in Fig. S1A shown, the amide I band (1660 cm−1 ), amide II (1540 cm−1 ) and amide III (1400 cm−1 ) band for CONH group appeared, which was due to the conjugation of DA to the MPA capped CuInS2 QDs. UV–vis absorption spectra (230–400 nm) of the DA-CuInS2 QDs were performed. As shown in Fig. S1B, the typical UV–vis absorption peak around 280 nm belonging to DA was observed in the DA-CuInS2 QDs, and not observed in the original MPA-capped CuInS2 QDs. It was further confirmed the presence of dopamine after the conjugation,
other possible interference on the fluorescence of DA-CuInS2 QDs, we respectively investigated the effect of urea, NH4 + , HCO3 − and urase on the fluorescence of DA-CuInS2 QDs. Fig. S3 shows the fluorescence intensity of DA-CuInS2 QDs with different concentration of urea, NH4 + , HCO3 − (in the range from 0 to 10 mmol/L) or urase (in the range from 0 to 1.25 mg/mL). It can be seen that urea, only NH4 + , HCO3 − or urase itself cannot induce obviously fluorescence quenching of the DA-CuInS2 QDs. So it was further confirmed that we could detect urea based on the fluorescence quenching of DACuInS2 QDs induced by OH− that was released from the hydrolysis of urea in the presence of urase. 3.3. Optimization for urea detection
3.2. The influence of pH on the fluorescence of DA-CuInS2 QDs The charge transfer interactions between fluorescent QDs and redox active dopamine have been extensively investigated [34,36]. In this work, we studied the effect of pH on the fluorescence properties of DA-CuInS2 QDs. As shown in Fig. 2, it could be seen that the fluorescence intensity of DA-CuInS2 QDs decreased with the increasing of the pH from 7.0 to 9.93, and when the pH increased to be 9.93, the fluorescence was almost entirely quenched. The significant fluorescence quenching was due to that the oxygen-catalytic oxidation of dopamine potentials was gradually reduced as the increased pH value from 7.0 to 9.93, and the equilibrium favored the transformation of dopamine to its oxidation product quinone at high pH. And the transfer of electrons from a photoexcited QDs to the lowest unoccupied molecular orbital of the proximal quinone acceptor would result in the fluorescence quenching of DA-CuInS2 QDs [34,36]. As shown in Fig. S2, under the acid conditions, the fluorescence intensity of DA-CuInS2 QDs did not exhibit significant variation with the decreasing pH value of the system. As well-known, the urea could release NH4 + , OH− and HCO3 − ions as products with urase as the catalyst. In order to exclude the
Fig. 1. The UV–vis absorption and fluorescence emission spectra of MPA-capped CuInS2 QDs (dash line) and DA-CuInS2 QDs (solid line).
As described in Eq. (1), the urea could release ionic product NH4 + , HCO3 − and OH− in the presence of urase. CO(NH2 )2 + 3H2 O = 2NH4 + + HCO3 − + OH−
(1)
As shown in Fig. 2, the fluorescence of DA-CuInS2 QDs would be quenched when pH of the system changed from neutral to alkalinity. Therefore, DA-CuInS2 QDs could be utilized as a fluorescence probe for the determination of urea with urase as catalyst. As wellknown that the urase plays a key role in the hydrolysis of urea. Therefore, choosing the appropriate concentration of urase could improve the sensitivity for urea detection by DA-CuInS2 QDs probe. In this work, the fluorescence intensity ratio F/F0 (F and F0 are the fluorescence intensity of DA-CuInS2 QDs with or without urea) of DA-CuInS2 QDs around 630 nm quenched by 2 mmol/L urea in the presence of different urase concentration (0.08–1.25 mg/mL) were studied, and the results were shown in Fig. 3. It can be seen that
Fig. 2. The fluorescence (FL) spectra of DA-CuInS2 QDs after the addition of different amounts of NaOH with the pH value of the system changed from 7.00 to 9.93 (pH = 7.00, 7.16, 7.27, 7.58, 7.96, 8.55, 8.95, 9.31, 9.70, 9.93). Inset: the plot of fluorescence quenching ratios of F/F0 at 630 nm versus the pH value of the system at room temperature.
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Fig. 3. The relationship between the fluorescence intensity ratio F/F0 of DA-CuInS2 QDs and the reaction time. F and F0 are the fluorescence intensity of DA-CuInS2 QDs with or without urea. Conditions: 2 mmol/L urea and various urase concentrations (0.08, 0.21, 0.42, 0.63, 1.25 mg/mL).
the fluorescence intensity ratio F/F0 of DA-CuInS2 QDs decreased slightly with the increasing of reaction time in the presence of low concentration of urase (0.08 or 0.21 mg/mL). When the urase concentration increased in the assay system (0.42–1.25 mg/mL), more dramatic changes of fluorescence intensity ratios F/F0 were observed. The gradually decreased fluorescence intensity ratios F/F0 with the increasing of reaction time clearly suggests that the urea was gradually decomposed to release OH− by catalysis of urase, and the increased concentrations of OH− in the assay system would result in the more obvious fluorescence quenching of DA-CuInS2 QDs. As Fig. 3 showed when the urase cencentration increased from 0.63 mg/mL to 1.25 mg/mL, the fluorescence intensity ratios (F/F0 ) after 30 min of enzyme hydrolysis almost reached the same. The results indicated that the urase concentration of 1.25 mg/mL was sufficient in this urea molecules hydrolysis system, and it was corresponding with previous reports [4,25]. In this paper, we investigated systematically the effect of the reaction temperature on this urea detection system, and the results were shown in Fig. S4. As curve a in Fig. S2 shown, the fluoresce intensity of DA-CuInS2 QDs decreased gradually with the increase of reaction temperature from 15 ◦ C to 40 ◦ C, which was due to that the increase of reaction temperature would increase the rate of oxidation and reduce the dopamine potential for oxidation and shifted the equilibrium toward the production of quinones [36,49–51]. Urase is an enzyme in organisms, and its activities increased gradually with the increase of temperature at a given range that was corresponding with fluorescence intensity changes of DA-CuInS2 QDs–urea–urase system (curve b). Considering the stability of DACuInS2 QDs, the activities of urase and being simple to operate, room temperature 20 ◦ C was chosen as the reaction temperature in our urea detection system. 3.4. Fluorescence detection of urea using the DA-CuInS2 QDs As shown in Fig. 4, the fluorescence of DA-CuInS2 QDs decreased as the urea concentration increased from 5 mol/L to 12 mmol/L in the presence of 1.25 mg/mL urase as catalyst. Fig. 4 inset describes the relationship between the fluorescence intensity ratio F/F0 (F and F0 are the fluorescence intensity of DA-CuInS2 QDs with or without urea) and the logarithm of urea concentration (0.005–12 mmol/L, logarithm value −2.30 to 1.08). It could be seen that when the concentration of urea was 12 mmol/L, the fluorescence intensity of DA-CuInS2 QDs would decrease to about 20% of original
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Fig. 4. The fluorescence (FL) spectra of DA-CuInS2 QDs upon the addition of different concentration of urea in the range of 0–12 mmol/L (0, 0.005, 0.020, 0.050, 0.10, 0.20, 0.50, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0, 12.0 mmol/L) in the presence of 1.25 mg/mL urase. Inset: the plot of fluorescence quenching ratios of F/F0 at 630 nm versus the concentration of urea at room temperature.
fluorescence intensity. The obviously fluorescence quenching induced by urea with urase as catalysts was due to the hydrolysis of urea, which released OH− to change the pH value of the system, and it favored easier transformation of dopamine to its oxidation product quinone. And then the transfer of electrons from QDs to the proximal quinone acceptor would result in the fluorescence quenching of DA-CuInS2 QDs. The proposed urea-biosensing system could effectively probe urea in the dynamic range from 0.2 to 6 mmol/L. As shown in Table 1, we compared the detecting limits and dynamic range of urea with various methodologies including potentiometry [10,52], amperometry [8], fluorescence measurements [4,25] and our proposed detection system. It can be seen that the proposed method offered a similar detecting limit and wide dynamic range for the detection of urea. Compared with previous reports about urea sensors, the DA-CuInS2 QD-based biosensor offered many advantages such as simple operations, low cost, without enzyme immobilization, good selectivity and sensitivity. 3.5. Effect of foreign substance In this paper, we studied the effect of some physiological ions and molecules on our proposed urea fluorescence detection system. It could be found that from Fig. S5A, the fluorescence intensity of DA-CuInS2 QDs with 1.25 mg/mL urase added by 2 mmol/L foreign substance K+ , Na+ , Ca2+ , Mg2+ , glucose (Glu), glucine (Gly), threonine (Thr) or lysine (lys) remained nearly constant, and only after the addition of urea, the fluorescence intensity showed obvious decreasing. The results indicated that the proposed urea detection system showed the high selectivity to urea. In Fig. S5B, we further investigated the fluorescence response of DA-CuInS2 QDs to 2 mmol/L urea with 1.25 mg/L urase in the presence of 2 mmol/L foreign substance K+ , Na+ , Ca2+ , Mg2+ , Glu, Gly, Thr or lys. It could be seen from Fig. S5B that even in the presence of 2 mmol/L interference, the DA-CuInS2 QDs sensor still work the same. The results showed that the proposed urea detection system could provide well ability of resisting interference from physiological ions and molecules. 3.6. Detection of urea in real samples In order to test the applicability of the proposed method, it was applied to determinate urea in human serum samples which spiked with different concentration of urea. The results obtained by
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Table 1 Comparison of performance of different urea sensors. Type
Sensing system
Detection limit (mmol/L)
Reference
Potentiometry
Poly(N-vinylcarbazole)/stearic acid Langmuir–Blodgett films Mercaptohydroquinone-modified gold electrode
10–68 0.2–5
5 0.2
[52] [10]
Amperometry
Urease + polyurethane–acrylate polytoluidine blue film
0.2–0.8
0.02
[8]
Fluorescence
Urea/MSA-CdSe/ZnS QDs Mercaptopropionic capped QDs–PAH-CaR Dopamine functionalized CuInS2 QDs
0.01–120 0.4–25 0.2–6
0.01 0.4 0.1
[25] [4] This work
standard addition method were shown in Table S1. The accuracy of the proposed method was evaluated by determining the recoveries of urea in real samples. It could be found that the average recoveries of urea in the real samples was in the range of 98–102% and the RSD was lower than 3.2%. The above results demonstrated that the potential applicability of the DA-CuInS2 QD-based fluorescence probe for the detection of urea in human serum samples. 4. Conclusion We have demonstrated the dopamine functionalized CuInS2 QDs that was sensitive to the pH values changes can be utilized as an effective fluorescence probe for the determination of urea. The fluorescence of the DA-CuInS2 QDs was quenched by urea in the range from 0.2 to 6 mmol/L with urase as the catalyst. The proposed method was successfully applied to the detection of urea in human serum sample with satisfactory results. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (Nos. 20875036 and 21075050) and the Science and Technology Development Project of Jilin Province, China (No. 20110334). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2013.09.056. References [1] B. Kovács, G. Nagy, R. Dombi, K. Tóth, Optical biosensor for urea with improved response time, Biosensors and Bioelectronics 18 (2003) 111–118. [2] J.M. Garrido, R. Mendez, J.M. Lema, Treatment of wastewaters from a formaldehyde-urea adhesives factory, Water Science and Technology 42 (2000) 293–300. [3] R. Doong, H.M. Shih, Array-based titanium dioxide biosensors for ratiometric determination of glucose, glutamate and urea, Biosensors and Bioelectronics 25 (2010) 1439–1446. [4] M.J. Ruedas-Rama, E.A.H. Hall, Analytical nanosphere sensors using quantum dot–enzyme conjugates for urea and creatinine, Analytical Chemistry 82 (2010) 9043–9049. [5] J.C. Chen, J.C. Chou, T.P. Sun, S.K. Hsiung, Portable urea biosensor based on the extended-gate field effect transistor, Sensors and Actuators: B Chemical 91 (2003) 180–186. [6] P.S. Chaudhari, A. Gokarna, M. Kulkarni, M.S. Karve, S.V. Bhoraskar, Porous silicon as an entrapping matrix for the immobilization of urease, Sensors and Actuators: B Chemical 107 (2005) 258–263. [7] W.Y. Lee, S.R. Kim, T.H. Kim, K.S. Lee, M.C. Shin, J.K. Park, Sol–gel-derived thickfilm conductometric biosensor for urea determination in serum, Analytica Chimica Acta 404 (2000) 195–203. [8] I. Vostiar, J. Tkac, E. Sturdik, P. Gemeiner, Amperometric urea biosensor based on urease and electropolymerized toluidine blue dye as a pH-sensitive redox probe, Bioelectrochemistry 56 (2002) 113–115. [9] A.P. Soldatkin, J. Montoriol, W. Sant, C. Martelet, N. Jaffrezic-Renault, A novel urea sensitive biosensor with extended dynamic range based on recombinant urease and ISFETs, Biosensors and Bioelectronics 19 (2003) 131–135. [10] F. Mizutani, S. Yabuki, Y. Sato, Voltammetric enzyme sensor for urea using mercaptohydroquinone-modified gold electrode as the base transducer, Biosensors and Bioelectronics 12 (1997) 321–328.
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Biographies Siyu Liu is currently carrying out his graduate work for his doctor degree under the guidance of Professor Su in Jilin University. His research focuses on the synthesis and functionalization of quantum dots and their application on sensors for metal ion and small molecules. Fanping Shi is currently carrying out her graduate work for her masters degree under the guidance of Professor Su in Jilin University. Her research focuses on the synthesis and functionalization of quantum dots and their application on sensors for metal ion and small molecules. Lu Chen received her bachelor degree in Institute of Chemistry Jilin University. Her research focuses on the application of quantum dots on sensors for small molecules. Xingguang Su is a professor at the Department of Analytical Chemistry at the College of Chemistry, Jilin University. She received her master degree from Jilin University (China) in 1992 and her doctor degree from Jilin University (China) in 1999. Her research focuses on the synthesis, characterization, functionalization and application of quantum dots and quantum dots-tagged microspheres in biomedicine.