Biosensors and Bioelectronics 22 (2007) 3283–3287
Piezoelectric urea biosensor based on immobilization of urease onto nanoporous alumina membranes Zhengpeng Yang, Shihui Si ∗ , Hongjuan Dai, Chunjing Zhang College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, China Received 24 October 2006; received in revised form 23 February 2007; accepted 2 March 2007 Available online 14 March 2007
Abstract The urease was immobilized onto nanoporous alumina membranes prepared by the two-step anodization method, and a novel piezoelectric urea sensing system with separated porous alumina/urease electrode has been developed through measuring the conductivity change of immobilized urease/urea reaction. The process of urease immobilization was optimized and the performance of the developed urea biosensor was evaluated. The obtained urea biosensor presented high-selectivity monitoring of urea, better reproducibility (S.D. = 0.02, n = 6), shorter response time (30 s), wider linear range (0.5 M to 3 mM), lower detection limit (0.2 M) and good long-term storage stability (with about 76% of the enzymatic activity retained after 30 days). The clinical analysis of the urea biosensor confirmed the feasibility of urea detection in urine samples. © 2007 Elsevier B.V. All rights reserved. Keywords: Urea; Activity; Urease immobilization; Urea biosensor; Nanoporous alumina membranes
1. Introduction The urea biosensor has been employed for urea determination in practical use due to its selectivity and operational simplicity. For the fabrication of an enzyme biosensor, an important step is to immobilize enzymes on the sensing surface (Arica et al., 1998; Lin and Brown, 1997; Jin and Brennan, 2002; Vinu et al., 2004; Ma et al., 2004; Gill et al., 1999; Lee et al., 2005). Usually the organic polymeric carriers, such as polyvinylalcohol (Miyata et al., 1997), polyethylenimine (Lakard et al., 2004) and polyvinylferrocenium (Kuralay et al., 2006) were employed for the immobilization of enzymes due to the presence of enough reactive functional groups. Compared with the organic polymeric carriers, the inorganic materials provide the better stability because of their thermal and mechanical stability and nontoxicity (Tischer and Wedekind, 1999). Various enzymes have been immobilized via inorganic materials, such as clay (Sanjay and Sugunan, 2005), porous silica (Lei et al., 2004), alumina powder (Reshmi et al., 2006). It is well known that porous alumina
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membranes with various dimensions could be easily fabricated via electrical anodization of high purity aluminum. These membranes have been used to prepare the nanoarrays and nanowires (Martin, 1994; Huczko, 2000; Metzger et al., 2000; Xiao et al., 2002), biosensors (Steinle et al., 2002; Kohli et al., 2004; Vlassiouk et al., 2005). It was found that the electrode-separated piezoelectric sensor (ESPS) was sensitive to conductive change of solution (Lamas-Ardisana and Costa-Garc´ıa, 2006; de Jesus et al., 2001). Compared to the conventional conductometry, the electrical double layer in the ESPS sensing system is eliminated owing to its very high working frequency (9 MHz). Meanwhile, it does not produce thermal effects for the biological materials because the detector cell is set in the feedback network of the oscillator and no obvious current flows through it. Moreover, the ESPS can detect a slight change in solution conductivity in the presence of an excess of foreign electrolyte, and the sensitivity and accuracy are better than those obtained in the absence of the foreign electrolyte (Shen et al., 1993). In the present study, the urease was immobilized onto nanoporous alumina membranes for the fabrication of ESPS urea biosensor, and the ESPS/FIA system was employed to monitor the enzyme reaction.
Z. Yang et al. / Biosensors and Bioelectronics 22 (2007) 3283–3287
2.3. Preparation of nanoporous alumina membranes
The nanoporous alumina membranes were prepared by the two-step anodization method described elsewhere (Masuda and Satoh, 1996; Masuda and Fukuda, 1995) (The preparation and characterization of nanoporous alumina membranes are shown in supplementary material).
High purity aluminum foils (99.99%, from VWR) were used to prepare nanoporous alumina membranes. Urease (EC 184.108.40.206, from jack bean, 50 U/mg) and glutaraldehyde were purchased from Sigma. Chitosan (degree of deacetylation: 88%, molecular weight: 210,000) was supplied by Hengsheng Biochemical Co., Qingdao, China. Urea and other chemicals were of analytical grade and used without further purification. Deionized (DI) water (resistivity of 18 M cm) was obtained from a Milli-Q system (Millipore Inc.), and used for rinsing and for makeup of all aqueous solutions.
2.2. Apparatus The construction of the ESPS/FIA is illustrated in Fig. 1. An overtone polished 9-MHz AT-cut quartz crystal (diameter 12 mm) with Au electrodes (diameter, 6 mm) on both sides was purchased from International Crystal Manufacturing (Oklahoma City, OK, USA). The quartz disc was flexibly fixed and sealed to the flow cell using silicon glue with the bare side of the quartz facing the liquid phase and the other side with the gold electrode in the air. A nanoporous alumina membrane with a geometrical area of 4 cm2 was used as the separated electrode. The distance between the separated electrode and the quartz crystal disc was set as 3 mm unless otherwise specified. The separated porous alumina electrode and gold electrode were connected to a home-made oscillator. The solution was introduced into flow cell by FIA setup. The oscillating frequency of the ESPS was monitored using a universal frequency counter (Iwatsu, Model SC-7201) attached to personal computer. The frequency drift of the measurement system was less than ±3 Hz in 20 min. Scanning electron micrographs (SEMs) of the home-made porous alumina membranes were taken using an Hitachi S520 scanning electron microscope (Hitachi Ltd., Tokyo, Japan) working at 20 kV.
2.4. Enzyme immobilization Methods of physical adsorption (procedure A), adsorption followed by cross-linking (procedure B) and cross-linking followed by chitosan coating (procedure C), were employed to immobilize enzyme (supplementary material). 2.5. Measurements of enzyme activity The activity mentioned in our study was the relative enzyme activity, which was estimated by measuring the frequency response of the urea biosensor in 1.0 mM urea solution (pH 7.5) at 35 ◦ C. Assuming that the enzyme activity was 100% at the maximum frequency response for each group of experiments, the value of enzyme activity was obtained from the ratio of the frequency response to the maximum frequency response. 3. Results and discussion 3.1. Immobilization of urease onto nanoporous alumina membranes Nanoporous alumina membranes have desired advantages for enzyme immobilization, such as relatively high surface area, high porosity, and high chemical, biological and mechanical stability (Darder et al., 2006). The penicillinaze enzyme has been successfully immobilized onto nano-structured anodic alumina membranes for the determination of penicillin (Takhistov, 2004). Herein, urease was immobilized onto nanoporous alumina membranes, and the frequency responses of ESPS/FIA detection system with the alumina electrode and the alumina/urease electrode were measured after the injection of 1.0 and 0.1 mM urea solutions (pH 7.5, 35 ◦ C). As seen in Fig. 2, the sensing system with alumina/urease electrode exhibited larger response to the same urea solution. The frequency decrease is attributed to the increasing conductivity. The catalytic reaction of urease-urea system can be described as follows: urease
NH2 CONH2 + 2H2 O −→ 2NH4 + + CO3 2−
Fig. 1. Setup of the ESPS/FIA detection system.
Thus, each initial uncharged urea molecule is changed into three ions, which lead to the increase of conductivity (namely larger frequency response) in the flow cell. In further study, the effects of pH, concentration of urease and temperature on urease immobilization onto nanoporous alumina membranes were performed (supplementary material, Fig. S2). The pH value of urease immobilization was optimized at 7.5, the enzyme loading was about 1.5 mg/cm2 . Considering that the isoelectric point of alumina is 9.1 (Brown et al., 1999) and that of
Z. Yang et al. / Biosensors and Bioelectronics 22 (2007) 3283–3287
Fig. 2. Frequency responses of ESPS/FIA detection system with the alumina electrode (a) and the alumina/urease electrode (b) after the injection of urea solutions. Experimental conditions: pH 7.5, 35 ◦ C and flow rate 8.5 mL/min.
Fig. 3. Reproducibility of urea biosensors with nanoporous alumina electrode soaked in 2.0 mg/mL urease solution (pH 7.5, 25 ◦ C) for 2.5 h, and followed by glutaraldehyde cross-linking for (A) 100, (B) 60, (C) 30 and (D) 0 min; (E) electrode (B) followed by chitosan coating.
urease is 5.5 (Koncki et al., 1992), the charge–charge interaction is presented between positive-charge alumina and negativecharge urease at pH 7.5. Our experimental results showed that the amount of adsorbed urease and the kinetic adsorption rate of urease on nanoporous alumina membranes were almost equal for various urease solutions (0.5, 1.0 and 2.0 mg/mL). It may be suggested that urease adsorption to nanoporous alumina membranes does not depend on diffusion of enzyme in solution but in pores of alumina (namely migration of urease into the inner pore channels of alumina membranes). The increasing temperature was favorable for enzyme immobilization because the migration of urease into the inner pores of alumina is an endothermic process. While for a long adsorption time (over 24 h), a almost equal activity of the adsorbed enzyme was observed at all temperatures (4–45 ◦ C), indicating that the amount of adsorbed enzyme was equal and the temperature would not affect the maximum immobilization of urease. This may be explained as follows: urease adsorption to porous alumina is a single molecular layer adsorption process; when the adsorption reaches saturation, further loading of urease will not prevented due to the high space hindrance in pores. Effect of pore dimensions (pore diameter and length) on urease immobilization was conducted in 2 mg/mL urease solution (pH 7.5) for 2.5 h at 25 ◦ C. It was found that the urease adsorbed to big pore possessed higher enzymatic catalytic activity. This phenomenon could be explained by the fact that nanoporous alumina membranes with big pore dimensions possesses the larger pore volume and specific surface area in comparison with that with smaller pore size. Thus, on one hand, the large pore volume is more favorable for enzyme immobilization than small pore volume; on the other hand, the large surface area increases the number of binding sites for the enzymes. The amount of enzymes immobilized in big pores reached 1.5 mg/cm2 , which was almost 12 times higher than that in small pores.
in a continuous manner and the obtained results were presented in Fig. 3. As compared to electrodes C and D, electrodes A, B and E exhibited better reproducibility due to the long cross-linking time, which hindered the leaching of enzyme from the pores in the reaction process. A decrease of enzyme activity was observed for a 100-min cross-linking time due to the high reticulation degree of the entrapped enzyme. It should be also noted that electrode E possessed good enzyme loading and the best reproducibility (S.D. = 0.02, n = 6, the sensing system with electrode E was employed in the further study). This could be explained by the fact that chitosan exhibits high permeability toward water, allows small molecule and ion transport, and shows a remarkable biocompatibility with enzyme (Krajewska, 2004).
3.2. Comparison of biosensor reproducibility
3.3.2. Response time, linear range, detection limit and selectivity The typical frequency response of the urea biosensor was conducted in 1.0 mM urea solution (pH 7.5) at 35 ◦ C. The steady-
Reproducibility of urea biosensors with five types of electrodes was evaluated by measuring the enzyme activity six times
3.3. Characteristics of the urea biosensor 3.3.1. Effect of temperature and pH on response of the urea biosensor Because biosensor response is known to be dependent on the enzyme activity (Kurakay et al., 2005), the effects of temperature and pH on urea biosensor response were examined by measuring enzyme activity (supplementary material, Fig. S3). In the temperature range of 15–45 ◦ C, the enzyme activity presented a linear increase with temperature up to 35 ◦ C, and then decreased due to the thermal denaturation of the enzyme caused by higher temperature. A maximum activity for free enzyme was found at pH 7.4, a higher pH inhibited enzyme activity due to the change of enzyme conformation at alkaline pH value. However, the high activity of the enzyme adsorbed on porous alumina was observed in a broader pH profile compared to free enzyme, suggesting that the immobilized enzyme system preserves good activity. This phenomenon could be attributed to the diffusional limitation or the secondary interactions between enzyme and the support (Arica and Hasirrci, 1993; He et al., 2000).
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Table 1 Interfering factors (f) of various interferents and selectivity (S) of the urea biosensor Interferent
10−4 M Glucose 10−4 M Creatinine 10−4 M Thiourea 10−4 M Glycine 10−5 M Serum 10−5 M Hg2+ 10−5 M Cu2+ 10−4 M NH4 + 10−4 M K+ 10−4 M Na+
1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
−190 −190 −190 −190 −190 −190 −190 −190 −190 −190
−188 −189 −186 −189 −184 −176 −180 −196 −194 −197
1.1 × 10−2 5.3 × 10−3 2.1 × 10−2 5.3 × 10−3 3.2 × 10−2 7.4 × 10−2 5.3 × 10−2 −3.2 × 10−2 −2.1 × 10−2 −3.7 × 10−2
0.989 0.995 0.979 0.995 0.968 0.926 0.947 1.032 1.021 1.037
a b c d
Frequency change for urea in the absence of the interferent. Frequency change for urea in the presence of the interferent. Interfering factor = (F0 − F)/F0. Selectivity of the urea biosensor = F/F0.
state response was reached within 30 s after injection of the urea solution. The response time was shorter than that reported in the literature (Cho and Huang, 1998; Singhal et al., 2002). The dynamic range of the urea biosensor was studied by monitoring the urea solution in the concentration range from 0.2 M to 12 mM. The experimental results showed that over the low concentration range (0.5 M to 3 mM), a good linearity was obtained. The linear regression equation was F (Hz) = −17.85–164.8[urea] (mM), R = 0.9996, n = 8. When the urea concentration was higher than 3 mM, the frequency response started to level off with the increasing of urea concentration. The sluggish frequency change for the higher urea concentration could be attributed to the kinetic restriction of the enzymatic reaction involved (Palmer, 1991). The detection limit of the urea biosensor was found to be about 0.2 M (S/N = 2), which was lower than that reported by other researchers (de Melo et al., 2002; Lee et al., 2000). The possible interferences of various biological, organic and inorganic species to the urea determination of the urea biosensor were investigated. As seen in Table 1, compared to the frequency response of the urea biosensor to standard urea solution, a slight change of frequency response was found for urea solutions with interferents. The interfering factors of various interferents are small (ca. 10−2 to 10−3 ) and the selectivity of the urea biosensor is extremely high (0.92–1.03) in the presence of these interfering species in urea solutions. Especially, what should be emphasized is that for urea detection, the piezoelectric urea biosensor avoided the interference of NH4 + in urea solution, which used to affect urea determination performed by other methods (Siegel et al., 1967; Cho and Huang, 1998). The high selectivity of the piezoelectric urea biosensor may make it possible to be applied widely for urea determination in the future.
4 ◦ C when not in use. It was found that the activity of enzyme decreased quickly in the first days and then became stable. The urea biosensor retained about 76% of its initial enzyme activity after 30 days. The loss of enzyme activity may be due to the denaturation of the immobilized enzyme during the long-term storage. To demonstrate the feasibility of the urea biosensor for clinical application, the recovery test of the urea biosensor was conducted by the standard addition method (supplementary material, Table S2). With five different additions of urea to the urine samples, the obtained recoveries ranged from 97.33 to 100.40%. The high recovery indicates that the urea biosensor is feasible for clinical analysis. Measurements of urea in real urine samples were performed by piezoelectric urea biosensor and clinical method. Good correlation was found between the two methods. Therefore, it is believed that a reliable piezoelectric urea biosensor has been obtained for urea determination in real urine sample in our study.
3.4. Storage stability and clinical application
This study was supported by the National Science Foundation of China (No.20475065).
The long-term storage stability of the urea biosensor was evaluated by monitoring activity of enzyme immobilized to porous alumina in 1.0 mM urea solution (pH 7.5) at 35 ◦ C. A total of nine measurements were taken for the urea biosensor in a month. The sensor was kept in 1.0 mM phosphate buffer solution at
4. Conclusions By immobilizing urease to nanoporous alumina membranes prepared by two-step anodization of Al sheet in oxalic acid solution, a novel piezoelectric urea biosensor with good reproducibility, wide linear range and high selectivity has been developed for the determination of urea. The detection of urea by ESPS urea biosensors is rapid and sensitive in the ESPS/FIA detection system, and the clinical application of the urea biosensor demonstrates the feasibility of urea determination in urine sample. Acknowledgement
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bios.2007.03.006.
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