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A new urea sensor based on combining the surface acoustic wave device with urease extracted from green soya bean and its application— determination of urea in human urine
Biosensors & Bioelectronics Vol. 11, No. 4, pp. 435-442, 1996
~) 1996 Elsevier Science Limited Printed in Great Britain. All rights reserved 0956-5663/96/$15.00
ELSEVIER ADVANCED TECHNOLOGY
A new urea sensor based on combining the surface acoustic w a v e device with urease extracted from green soya bean and its application-determination of urea in human urine Dezhong Liu, Kang Chen, Kai Ge, Lihua Nie & Shouzhuo Yao* Department of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China Tel: [86] (731) 8822648 Fax: [86] (731) 8824287 (Received 6 September 1994; revised 2 June 1995; accepted 19 June 1995)
Abstract: The urea sensor was prepared by combining a surface acoustic wave
(SAW) device, in which a SAW resonator operating at 61 MHz and a pair of parallel electrodes were used in series, with urease extracted from green soya bean. The Michaelis constant and maximum reaction rate of the urease were estimated as 2.14 mM and 27.18 kHz min -~, respectively, at pH 7.0 and 25.0°C. Influences of pH, temperature and effectors on the response properties of the SAW urea sensor were investigated. Recovery of the sensor ranged from 95 to 105% and the detection limit of urea was 1.0/zg ml -~ (1.7 x 10-5 M). The proposed sensor has been successfully applied to the rapid determination of urea in human urine samples. The results are consistent with the reported values and also support the clinical diagnosis. Keywords: surface acoustic wave, urea sensor, urease, urine
INTRODUCTION A surface acoustic wave (SAW) device is a precise generator and detector of acoustic waves which oscillates at a characteristic resonant frequency when connected to a feedback circuit with a radio frequency amplifier. In most chemical sensor applications (D'Amico et al., 1982; Wohltjen, 1984; Ricco et al., 1985; Ballantine et al., 1986), the SAW device is coated with a special polymer which absorbs the chemical
* To whom correspondence should be addressed.
substance of interest. The SAW velocity changes due to the absorbed chemical substance can result in the alternation of the mass load, the elastic stress and the conductivity of the polymer coating (Heckl et al., 1990). Chemical concentration can therefore be tested by monitoring the oscillation frequency. However, a large energy loss of the Rayleigh wave in the liquid phase limits the SAW device sensing in the liquid phase (Calabrese et al., 1987). A new type of SAW sensor device utilizing a 61 MHz SAW resonator and a pair of parallel electrodes in series has recently been introduced to our laboratory (Yao et al., 1994). This device 435
Dezhong Liu et al.
Biosensors & Bioelectronics
is based on the principle that only when the sum of the phase angles of a loop circuit containing resistive, capacitive and inductive elements in series with an amplifier and a SAW device is a multiple of 27r rad and the loop gain is greater than 1 will oscillation be generated and supported. If any element in the circuit imparts a phase delay change, the oscillation frequency will change. Therefore, it can respond to any changes in the physical/chemical properties of the medium between two electrodes as they cause variations in the loop parameters. By analyzing the frequency shift response much information about the liquid system can be obtained. This device can be applied to the study of the liquid phase, for which it was previously considered that SAW devices could not be used (Calabrese et al., 1987). As proposed earlier (Yao et al., 1994) a linear relationship exists between the frequency shift (AF) and the change of electrolyte conductivity (AK) and the permittivity (Ae) of the solution: AF = a AK + b
(1)
AF = cAc + d
(2)
where a, b, c and d are constants depending on the SAW device, the circuit used and other experimental conditions. The effects of the density and viscosity of the solution were also investigated. The above two parameters have little effect on the frequency of the sensor system, but do affect the capacitance and conductance of the solution since the SAW resonator is not in contact with the solution and its frequency should depend only on the electrolyte conductivity and permittivity. In this work, no significant changes of the permittivity and viscosity took place. Only a change of the concentration of electrolyte solution will cause a frequency response of the SAW urea sensor system. We also found that in a certain conductivity range (corresponding to KCI concentration up to 0.02-0.03 mol 1-1), the higher the conductivity is, the higher the sensitivity of the sensor system. This advantage makes the sensor system more attractive than the normal a.c. conductometry and a.c. impedance measurement in comparison with the normal conductometry. In addition, temperature drift of the sensor system is very small, only - 1 0 Hz °C -1, when applied in the liquid phase. Urea is the chief nitrogenous end-product of 436
protein catabolism in humans and other mammals. Determination of urea concentration in urine and blood is important for the diagnosis of liver and kidney diseases. There are several methods for urea determination based on the use of urease: amperometry (Pandey & Mishra, 1988); potentiometry (Abdulla et al., 1989; Campanella et al., 1990); fiber-optic sensor (Xie et al., 1991); and conductometry (Duffy et al., 1988; Cullen et al., 1990). Shiokawa and coworkers (1993, 1994) have reported a shear horizontal mode surface acoustic wave (SH-SAW) biosensor for the liquid detection of two kinds of enzymes, urease and cholinesterase, in which the detection principle is the acoustoelectric interaction due to the pH change in the enzyme reaction. This sensor can respond to the change of viscosity, massloading, permittivity and conductivity only in the adjacent liquid of the piezoelectric materials. In the urea/urease reaction system: H2NCONH2 + 3H20
Urease
> 2NH4~
+ HCOf + OH-
(3)
the initially uncharged substrate (urea) is hydrolyzed to yield four charge-beating species. Conductometry allowed the determination of urea at the 30 ~mol 1-1 level in 0.1 mmol 1-1 Tris-HCl buffer (Jespersen, 1975), but the medium composition (especially the choice of buffer and its ionic strength) and the temperature must be carefully controlled. It was reported that a temperature change of 0.01°C led to an average conductivity variation of about 0.02%, greater than the amplitude of the hydrodynamic and electronic background noise (Duffy et al., 1988). Other methods suffered from poor precision or low sensitivity. Being different from the principle of SH-SAW sensor reported by Shiokawa and coworkers (1993, 1994), the present SAW urea sensor responds predominantly to the change of conductivity in the bulk solution and can therefore be applied to the highly sensitive detection of some of the enzymatic reactions. Such a sensor is advantageous in its simplicity in construction, high sensitivity and low noise level (the typical noise level is less than 2 Hz). In this work, the kinetic parameters of the urease were estimated and the pH, temperature and effectors on the sensor were also investigated. Results of the determination of urea in urine samples are reported.
Biosensors & Bioelectronics
A new urea sensor
EXPERIMENTAL
Reagents Urease (EC 3.5.1.5, from jack bean, 1.7 EU mg-1), purchased from BDH Chemicals Ltd. (Poole, UK), was used to evaluate the specific activity of urease from green soya bean. Stocking solution of urease (5 mg m1-1) was prepared with 20% glycerol-0.02% sodium azide and was stored in a refrigerator before use. Human seroalbumin was obtained from Shanghai Biochemical Products Institute. A control urine sample, which was composed of 5.0 mg ml glucose, 1.0 mg ml l human seroalbumin and 5.0 mg ml -] urea, was obtained from the clinic hospital. Human urine samples were collected freshly from the healthy or from patients; it was the first urine of the early morning. The samples were detected immediately without any treatment or delay. The working buffer was 1.0 mM glycine-l-0 mM EDTA with pH 7.0. All other chemicals were obtained commercially and were of analytical reagent grade. All solutions were prepared with double distilled water.
Apparatus The 61 MHz one-port resonators used in this study were originally manufactured by Zhuzhou Radio Factory (Hunan), in y,z-cut LiNbO3 crystal with aluminum metallization, and mounted on round two-pin To-5 headers with epoxy and gold wirebonds. The aluminum interdigital transducers (IDTs) were 13,000/~ thick. The resonators were designed with an acoustic aperture and a path length of several wavelengths. On the center of each separate LiNbO3 crystal chip, there are 20 pairs of IDTs with 500 reflectors placed on each side of them. The nominal insertion loss of the resonators is 6.8 dB. The whole device was sealed for protection against the atmosphere with an epoxide. A schematic diagram of the experimental setup is shown in Fig. 1. Cpc is a parallel capacitor. In all experiments, the detection cell was placed in an air-bath thermostating equipment (homemade) in which the temperature was monitored and controlled by a model WMZK-01 Temperature Controller (Medical Instrument Co., Shanghai). The platinum electrodes were placed parallel in a detection cell (its cell constant was 1.04 cm 1). Not being in contact with the
OSCILLATOR CIRCUITRY
-
-
FREQUENCY COUNTER
F [
~|
COMPUTER
PLOTTER
SBN II RESONRTOR
DETECTION CELL
Fig. 1. Schematic diagram of the experimental setup. Cpc is a parallel capacitor.
solution, such a device should be electrically connected as close as possible to the pair of parallel platinum electrodes set in a 10 ml detection cell. The solution in the cell was stirred with a magnetic stirrer at a constant stirring rate. The oscillation frequency of the SAW resonator was measured by using a model SC7201 Iwatsu Universal Counter at a resolution of 1 Hz, and data were collected at 6 points min -]. The pH of the working buffer was measured by a model EA940 Orion ion analyzer using a calibrated glass electrode. A personal computer was used for data processing. A d.c. power of 8.0 V was supplied by an adjustable dual track d.c. power supply.
Preparation of urease extract Urease from green soya bean was extracted by a simple procedure (Torrey, 1983). No further purification procedures were needed. At first, green soya bean was finely ground using an electric smasher and was screened to pass a 60mesh sieve, then 5.00 g of the powder was weighed and transferred to a 100 ml Erlenmeyer flask with a stopper. Fifty milliliters of organic extracting solvent were added to this vessel. The mixture was shaken on an oscillating machine for 30 min at room temperature and kept in a refrigerator at 4°C for 4 h. The solution was then centrifuged at 2000 g for 10 min. The urease solution was obtained after phase separation. It was stored in a brown flask and kept in a refrigerator before the experiment. The crude extract can be used directly in the measurements below. The urease concentration in this extract was estimated as 25-0 EU ml-~ by comparing its kinetic response with the commercial urease at 2-0 mg m1-1 urea. Experiments have shown that 437
Dezhong Liu et al.
the activity of urease extract can remain steady for about 4 weeks.
Biosensors & Bioelectronics
G.0 /'4
5.0 Frequency response measurements
\
b
.,4.0 4¢.
The whole setup must be turned on for at least 30 min in order to guarantee stabilization before the experiment. The air bath was thermostated at 25 - 0.2°C. A typical procedure was the following: 0.50 ml urease solution and 9.50 ml working buffer were transferred to the detection cell. The temperature of the detection cell was thermostated at 25.0 --- 0.2°C and the solution was stirred by a magnetic stirrer. Frequency drift was recorded prior to adding urea solution, then the steady frequency was recorded (Fo). After a definite volume of standard urea solution was added with a 50 ~1 syringe, the enzyme-catalyzed reaction was started and the oscillation frequency changed with time. Frequency (F) was recorded with time and the frequency shift (AF) was calculated by subtracting Fo from F. The calibration curve was obtained by measurement of the sensor responses to different amounts of the control urine sample. Only a 5.0/.d human urine sample was needed for urea measurement and the urea concentration was estimated by the calibration method.
RESULTS AND DISCUSSION
C
.c 3.0 ~2.0 cO
:
d
1.0
~ 0.0 I
I
1.o 21o 3.0 Time / min
,
I
5.0
Fig. 2. Frequency response of the SAW urea sensor for different concentrations of urease from green soya bean in the reaction system: (a) 1.75; (b) 1.25; (c) 0.75; (d) 0.25 EU m1-1. 0.10 mg m1-1 urea was used in this procedure. 7,0-
~ S.O
c
5.0
cl
~_ 4,0 c-
3,0
~.~ ~.~ I
h
I
,
I
010 1,0 2.0 3.0 4.0 5,0 G,~ Typical kinetic responses for different amounts of urease Typical responses of the SAW urea sensor to standard urea under the condition of different concentrations of urease are shown in Fig. 2. The substrate urea concentration was 0.10 mg m1-1 in this experiment. It can be seen that frequency response increases with the increase of urease concentration, i.e. the reaction rate increases with increasing urease content. The response curve of the SAW urea sensor is analogous to normal enzymatic reaction (Skoog et al., 1988). Selection of extracting solvent Figure 3 has shown the frequency response for the following four extracting solvents: 20% glycerol, 30% ethanol, 32% acetone (all in volume ratio) and water. It can be seen that 438
Time
(min)
Fig. 3. Frequency responses for different kinds of extracting solvent used in the preparation of urease from green soya bean: (a) 20% glycerol; (b) 30% ethanol; (c) 32% acetone; (d) water. 0.20mg m1-1 urea and 1.25 EUm1-1 urease were used in this experiment.
three kinds of organic solvents possess almost the same extracting ability. There is a lower urease activity for water as the extracting solvent than for the other solvents. Twenty percent glycerol was the best extract-solvent and was selected because of its protective effect on urease activity. Effects of pH and temperature Enzymes are very sensitive to changes of pH and function best over a very limited range with a
Biosensors & Bioelectronics
A new urea sensor
a considerable effect on the frequency response (urease activity). Within the range of 34--38°C, the temperature influence can be neglected and the sensor system yields the greatest sensitivity. A temperature of 25°C was selected in our experiment by considering the measurement conditions.
2, 6am ¢,J
¢ > ~ 40 Kinetic
% --
parameters
20
r-t
,
o ,0
I
~
I
6.g
,
?.0
I
,
l°J
8.0
£.0
10.0
pH
Fig. 4. Effect of p H on urease activity. 1.25 EU ml -~ urease extracted from green soya bean and 50 Ixg m1-1 urea were selected in this measurement.
definite pH optimum. The effects of pH are due to changes in the ionic state of the amino acid residues of the enzyme, influencing the binding of the substrate with enzyme and affecting the rate of reaction. Over a narrow pH range, these effects will be reversible. Variations in the pH and temperature of the buffer solution also produce ion strength variations. Calculated amounts of NaCI solution were added to the buffer in such a way that, for each experimental point, a constant value of ionic strength was maintained. Figure 4 shows the effect of the pH of the glycine buffer (1-0 mM) on the SAW urea sensor response over the pH ranges 5.5-9.3. It can be seen that the sensor gives the maximum sensitivity at pH 7.0. Temperature greatly affects the activity of the enzyme. Figure 5 shows that the temperature has
Kinetic parameters of urease to urea depend not only upon its source, reaction pH and temperature, but also upon the type and concentration of buffer. For jack bean urease, the value of the Michaelis constant Km ranges between 1 and 5 mM, according to the experimental conditions (Lynn & Yankwick, 1964; Lynn, 1967). In general, the amino buffer (e.g. glycine) results in a greater enzyme activity than does the simple inorganic buffer such as phosphate or carbonate. In this experiment, 1.0 mM glycine buffer (pH 7.0) and 25.0°C were selected. The Km and maximum rate for the urease from green soya bean were estimated as 2.14 mM and 27.18 kHz min -1 separately, by using Lineweaver-Burk analysis corresponding to substrate concentrations ranging from 1.0 to 20 tzg ml -~. Calibration
Figure 6 depicts typical responses of the SAW urea sensor to standard urea solution in different concentrations. There is a linear correlation
N "r
.,. t00
curves and recovery
\ w
80
7.0
7.g
6.0-
~.0 ~"
5.0
5.8 --
4.0
4.0
3.0
3.~
20 •
2.0
"3
c"
.~
60
I.I
~>
40
--
20
r-
g, 1.0
I N \
[.0 3
t.~
rv,
I
0
tO
,
I
~
l
,
I
~
i
20 30 40 50 T e m p e r a t u r e / °C
0
,
GO
Fig. 5. Effect o f temperature on urease activity. The experimental conditions were the same as in Fig. 4.
10
20 CUrea]
30
40
50
(~g/mL)
Fig. 6. Calibration curves for determination of urea. 1.25 E U m1-1 urease from green soya bean was used in the assay of urea.
439
Dezhong Liu et al.
Biosensors & Bioelectronics
AF = 233.7 [Urea] + 10, n = 8, r = 0.9982
Effect of activators and inhibitors
(4) where the urea concentration is expressed in /xg m1-1 and AF in Hz. The detection limit of the SAW urea sensor as 1.0/~g m1-1 (1.7 × 10 -5 mol 1-1). As derived by Michaelis and Menten (see Palme, 1981) the initial rate (Vo) is given by k[E][Urea] Vo - [Urea] + Km
(5)
where k is the rate constant relating to product formation, [E] is the concentration of urease, and Km is the Michaelis constant. Hence, it is evident that Vo is directly proportional to the content of the enzyme when [Urea] ~ Km and at constant concentration of substrate, so the urea concentration can be detected by using the kinetic method. A correlation of the initial rate vs. urea concentration was presented: Vo = 181.7 [Urea] + 19,
n = 8, r = 0.9990
(6) where the urea concentration is expressed in /zg ml -x and Vo in Hz min -1. The linearity ranges up to 17.5/zg m1-1. The kinetic method can decrease the measurement time but it has a lower sensitivity and a narrower linearity than the fixedtime method by comparing Eqs.(4) and (6). So the fixed-time method was employed for the determination of urea concentration. Recovery experiments have been carried out with normal urine spiked with known urea concentrations. The recovery ranged from 95 to 105% (Table 1).
TABLE 1 Analytical recovery*. Urea (l~g rn1-1) Added
Found
1.00 2.50 5.00 10.0 15.0 20-0
0.95 2.61 4.91 9-62 15.4 19.6
Recovery (%)
95-0 104.4 98.2 96-2 102.3 98.3
Mean (%)
RSD (%)
99.1
3.70
*For experimental conditions see legend to Fig. 6. All results were calculated by the calibration equation. 440
Effects of interferents on frequency response of the SAW urea sensor are shown in Table 2. Results have shown that an amount of activator such as the amino acid glycine can increase the rate of urease reaction in the test solution; there were no observable influences on rate of urease reaction for sodium chloride, glucose, starch, human serumalbumin. Inhibitors such as heavy metal ions (Pb 2÷, Hg 2÷, Ag ÷) decrease the reaction rate and Hg 2÷ and Ag ÷ seriously inhibited urease reaction. Figure 7 shows the frequency-shift response in the presence of different concentrations of inhibitor Hg z+. Results show that the urease reaction rate decreases with increasing content of Hg 2÷ in the test solution, and Hg 2÷ is an effective inhibitor for urease. This property may be used as a method for the determination of mercury(II). Inhibitor-like thiourea structurally resembles the urea and may be bound by the urease, but cannot be converted to the products. Figure 8 shows the influence of thiourea on the relative initial rate of the reaction (i.e. relative activity (RA) of urease). The plot is linear, ranging from 0 to 6.0 mM thiourea: R A (%) = - 1 0 . 4 [Thiourea] + 98.9,
T A B L E 2 Effects of interferents on frequency response of the SAW urea sensor*. lnterferents
None Glucose Soluble starch Human serumalbumin Sodium chloride Zinc chloride Ferric chloride Ferrous sulfate Lead nitrate Cadmium nitrate Silver nitrate Mercury nitrate Thiourea Glycine
Concentration
4.0 mg m1-1 4.0 mg m1-1 2-0 mg m1-1 1-0 mM 0-10 mM 0-10 mM 0.10 mM 0-10 mM 0.10 mM 0.05 mM 1.00/zM 1.00 mM 1.00 mM
Relative response (%) 100 97 98 97 99 95 94 97 95 94 80 85 94 103
*The concentration of urea was 50/~g m1-1 (0.83 mM). For experimental conditions see Experimental section.
A new urea sensor
Biosensors & Bioelectronics
TABLE 3 Assay of urea content in human urine samples.
8.0
Sex ~ \
S.0
b
'*'4.0
Clinical diagnostic results
Tested Normal value value (Ye et al., 1989)
c
.£:
I 0.0
~
I 1.0
1
[ 2.0
T~me
,
, 3.0
I
I 4.0
Content of urea (mg m1-1)
1
/ min
Fig, 7. Responses of the urea sensor to standard urea solutions (50 I~g m1-1) with inhibitor Hg e÷ in different concentrations: (a) O; (b) 1.0," (c) 2.5; (d) 5.0; (e) 7.5 tzM.
Female Female Male Male Male Male Male Female Female Male Male
Healthy Healthy Healthy Healthy Healthy Healthy Chronic nephritis Chronic nephritis Nephrotic syndrome Chronic nephritis Nephrotic syndrome
12.5 12.9 19.8 16.3 12.4 16.7 9.32 8-95 9.90 9.93 7.36
10-20
•~" 1 0 0
patients with liver or kidney diseases, the urea content in urine is lower than the normal value. The tested results reflect the facts and support the clinical diagnosis. The above-mentioned differences of urea content in urine between tested and normal values for renal disease patients have been explained pathologically (Harrington & Zimmerman, 1982), therefore analysis of urea content in urine can deliver important diagnostic evidence.
> 60
_
O
c[
40
) 20
-
a~
0.0
1.0 2.[3 3.0 CThiourea]
4.0 5.0 ( mM )
6.0
Fig. 8. Relative activity of urease as a function of the concentration of inhibitor thiourea. The experimental conditions were the same as in Fig. 4.
n = 7, r = -0.996
(7)
Determination of urea in human urine
Urea concentrations of 11 urine samples were then determined based on the above calibration equation and 5.0/xl of urine samples was used for all assays. The results are summarized in Table 3. The clinical analysis of urea has shown: (1) for healthy adults, the urea concentrations coincide with the literature range (Ye et al., 1989); (2) for
CONCLUSIONS In conclusion, the proposed SAW urea sensor, combining the sensitivity of the SAW sensor device in a relative high concentration electrolyte and the specificity of urease, is well-adapted for routine urea assay of small urine samples. This method is simple and sensitive and provides a uniquely advantageous specific assay of urea.
ACKNOWLEDGEMENTS This work was financially supported by the National Natural Science Foundation of China and Education Commission Fund of China. 441
Dezhong Liu et al. REFERENCES Abdulla, H.M., Greenway, G.M., Platt, A.E. & Fielden, P.R. (1989). Analyst, 114, 1575. Ballantine, D.S., Rose, S.L., Grate, J.W. & Wohltjen, H. (1986). Anal. Chem., 58, 3058. Calabrese, G.S., Wohltjen, H. & Roy, M.K. (1987). Anal. Chem., 59, 833. Campanella, L., Sammartino, M.P. & Tomassetti, M. (1990). Analyst, 115, 827. Cullen, D.C., Sethi, R.S. & Lowe, C.R. (1990). Anal. Chim. Acta, 231, 33. D'Amico A., Palma, A. & Verona, E. (1982). Appl. Phys. Lett., 41, 300. Duffy, P., Mealet, C., Wallaeh, J.M. & Fombon, J.J. (1988). Anal. Chim. Acta, 211, 205. Harrington, A.R. & Zimmerman, S.W. (1982). Renal Pathophysiology. John Wiley & Sons, New York. Heckl, W.M., Marassi, F.M., Kallury, K.M.R., Stone, D.C. & Thompson, M. (1990). Anal. Chem., 62, 32. Jespersen, N.D. (1975). J. Am. Chem. Soc., 97, 1662. Kondoh, J., Matsui Y. & Shiokawa, S. (1993). Jpn. J. Appl. Phys., 32, 2376. Kondoh, J., Matsui, Y., Shiokawa S. & Wlodarski, W.B. (1994). Sensors & Actuators B, 20, 199.
442
Biosensors & Bioelectronics Lynn, K.R. (1967). Biochim. Biophys. Acta, 146, 205. Lynn, K.R. & Yankwick, P.E. (1964). Biochim. Biophys. Acta, 81, 333. Palme, T. (1981). Understanding Enzymes. Ellis Horwood Ltd., Chichester. Pandey, P.C. & Mishra, A.P. (1988). Analyst, 113, 329. Ricco, A.J., Martin, S.J. & Zipperian, T.E. (1985). Sensors & Actuators, 8, 319. Skoog, D.A., West D.M. & Holler, F.J. (1988). Fundamentals of Analytical Chemistry. Saunders College Publishing, New York, 5 Edn. p. 587. Torrey, S. (ed.) (1983). Enzyme Technology (Preparation, Purification, Stabilization, Immobilization): Recent Advances. Noyes Data Corporation, New Jersey, pp. 198-200. Wohltjen, H. (1984). Sensors & Actuators, 5, 307. Xie, X., Suleiman, A.A. & Guilbault, G.G. (1991). Talanta, 38, 1197. Yao, S., Chen, K., Liu, D. & Nie, L. (1994). Anal. Chim. Acta, 294, 311. Yao, S., Chen, K., Nan, F., Shen, D. & Nie L. (1994). Anal. Chim. Acta, 287, 65. Ye, Y.W., Li, J.Z. & Wang, Y.S. (1989). Clinic Detection and Diagnosis. People's Hygiene Press, Beijing, p. 2331 (in Chinese).
~) 1996 Elsevier Science Limited Printed in Great Britain. All rights reserved 0956-5663/96/$15.00
ELSEVIER ADVANCED TECHNOLOGY
A new urea sensor based on combining the surface acoustic w a v e device with urease extracted from green soya bean and its application-determination of urea in human urine Dezhong Liu, Kang Chen, Kai Ge, Lihua Nie & Shouzhuo Yao* Department of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China Tel: [86] (731) 8822648 Fax: [86] (731) 8824287 (Received 6 September 1994; revised 2 June 1995; accepted 19 June 1995)
Abstract: The urea sensor was prepared by combining a surface acoustic wave
(SAW) device, in which a SAW resonator operating at 61 MHz and a pair of parallel electrodes were used in series, with urease extracted from green soya bean. The Michaelis constant and maximum reaction rate of the urease were estimated as 2.14 mM and 27.18 kHz min -~, respectively, at pH 7.0 and 25.0°C. Influences of pH, temperature and effectors on the response properties of the SAW urea sensor were investigated. Recovery of the sensor ranged from 95 to 105% and the detection limit of urea was 1.0/zg ml -~ (1.7 x 10-5 M). The proposed sensor has been successfully applied to the rapid determination of urea in human urine samples. The results are consistent with the reported values and also support the clinical diagnosis. Keywords: surface acoustic wave, urea sensor, urease, urine
INTRODUCTION A surface acoustic wave (SAW) device is a precise generator and detector of acoustic waves which oscillates at a characteristic resonant frequency when connected to a feedback circuit with a radio frequency amplifier. In most chemical sensor applications (D'Amico et al., 1982; Wohltjen, 1984; Ricco et al., 1985; Ballantine et al., 1986), the SAW device is coated with a special polymer which absorbs the chemical
* To whom correspondence should be addressed.
substance of interest. The SAW velocity changes due to the absorbed chemical substance can result in the alternation of the mass load, the elastic stress and the conductivity of the polymer coating (Heckl et al., 1990). Chemical concentration can therefore be tested by monitoring the oscillation frequency. However, a large energy loss of the Rayleigh wave in the liquid phase limits the SAW device sensing in the liquid phase (Calabrese et al., 1987). A new type of SAW sensor device utilizing a 61 MHz SAW resonator and a pair of parallel electrodes in series has recently been introduced to our laboratory (Yao et al., 1994). This device 435
Dezhong Liu et al.
Biosensors & Bioelectronics
is based on the principle that only when the sum of the phase angles of a loop circuit containing resistive, capacitive and inductive elements in series with an amplifier and a SAW device is a multiple of 27r rad and the loop gain is greater than 1 will oscillation be generated and supported. If any element in the circuit imparts a phase delay change, the oscillation frequency will change. Therefore, it can respond to any changes in the physical/chemical properties of the medium between two electrodes as they cause variations in the loop parameters. By analyzing the frequency shift response much information about the liquid system can be obtained. This device can be applied to the study of the liquid phase, for which it was previously considered that SAW devices could not be used (Calabrese et al., 1987). As proposed earlier (Yao et al., 1994) a linear relationship exists between the frequency shift (AF) and the change of electrolyte conductivity (AK) and the permittivity (Ae) of the solution: AF = a AK + b
(1)
AF = cAc + d
(2)
where a, b, c and d are constants depending on the SAW device, the circuit used and other experimental conditions. The effects of the density and viscosity of the solution were also investigated. The above two parameters have little effect on the frequency of the sensor system, but do affect the capacitance and conductance of the solution since the SAW resonator is not in contact with the solution and its frequency should depend only on the electrolyte conductivity and permittivity. In this work, no significant changes of the permittivity and viscosity took place. Only a change of the concentration of electrolyte solution will cause a frequency response of the SAW urea sensor system. We also found that in a certain conductivity range (corresponding to KCI concentration up to 0.02-0.03 mol 1-1), the higher the conductivity is, the higher the sensitivity of the sensor system. This advantage makes the sensor system more attractive than the normal a.c. conductometry and a.c. impedance measurement in comparison with the normal conductometry. In addition, temperature drift of the sensor system is very small, only - 1 0 Hz °C -1, when applied in the liquid phase. Urea is the chief nitrogenous end-product of 436
protein catabolism in humans and other mammals. Determination of urea concentration in urine and blood is important for the diagnosis of liver and kidney diseases. There are several methods for urea determination based on the use of urease: amperometry (Pandey & Mishra, 1988); potentiometry (Abdulla et al., 1989; Campanella et al., 1990); fiber-optic sensor (Xie et al., 1991); and conductometry (Duffy et al., 1988; Cullen et al., 1990). Shiokawa and coworkers (1993, 1994) have reported a shear horizontal mode surface acoustic wave (SH-SAW) biosensor for the liquid detection of two kinds of enzymes, urease and cholinesterase, in which the detection principle is the acoustoelectric interaction due to the pH change in the enzyme reaction. This sensor can respond to the change of viscosity, massloading, permittivity and conductivity only in the adjacent liquid of the piezoelectric materials. In the urea/urease reaction system: H2NCONH2 + 3H20
Urease
> 2NH4~
+ HCOf + OH-
(3)
the initially uncharged substrate (urea) is hydrolyzed to yield four charge-beating species. Conductometry allowed the determination of urea at the 30 ~mol 1-1 level in 0.1 mmol 1-1 Tris-HCl buffer (Jespersen, 1975), but the medium composition (especially the choice of buffer and its ionic strength) and the temperature must be carefully controlled. It was reported that a temperature change of 0.01°C led to an average conductivity variation of about 0.02%, greater than the amplitude of the hydrodynamic and electronic background noise (Duffy et al., 1988). Other methods suffered from poor precision or low sensitivity. Being different from the principle of SH-SAW sensor reported by Shiokawa and coworkers (1993, 1994), the present SAW urea sensor responds predominantly to the change of conductivity in the bulk solution and can therefore be applied to the highly sensitive detection of some of the enzymatic reactions. Such a sensor is advantageous in its simplicity in construction, high sensitivity and low noise level (the typical noise level is less than 2 Hz). In this work, the kinetic parameters of the urease were estimated and the pH, temperature and effectors on the sensor were also investigated. Results of the determination of urea in urine samples are reported.
Biosensors & Bioelectronics
A new urea sensor
EXPERIMENTAL
Reagents Urease (EC 3.5.1.5, from jack bean, 1.7 EU mg-1), purchased from BDH Chemicals Ltd. (Poole, UK), was used to evaluate the specific activity of urease from green soya bean. Stocking solution of urease (5 mg m1-1) was prepared with 20% glycerol-0.02% sodium azide and was stored in a refrigerator before use. Human seroalbumin was obtained from Shanghai Biochemical Products Institute. A control urine sample, which was composed of 5.0 mg ml glucose, 1.0 mg ml l human seroalbumin and 5.0 mg ml -] urea, was obtained from the clinic hospital. Human urine samples were collected freshly from the healthy or from patients; it was the first urine of the early morning. The samples were detected immediately without any treatment or delay. The working buffer was 1.0 mM glycine-l-0 mM EDTA with pH 7.0. All other chemicals were obtained commercially and were of analytical reagent grade. All solutions were prepared with double distilled water.
Apparatus The 61 MHz one-port resonators used in this study were originally manufactured by Zhuzhou Radio Factory (Hunan), in y,z-cut LiNbO3 crystal with aluminum metallization, and mounted on round two-pin To-5 headers with epoxy and gold wirebonds. The aluminum interdigital transducers (IDTs) were 13,000/~ thick. The resonators were designed with an acoustic aperture and a path length of several wavelengths. On the center of each separate LiNbO3 crystal chip, there are 20 pairs of IDTs with 500 reflectors placed on each side of them. The nominal insertion loss of the resonators is 6.8 dB. The whole device was sealed for protection against the atmosphere with an epoxide. A schematic diagram of the experimental setup is shown in Fig. 1. Cpc is a parallel capacitor. In all experiments, the detection cell was placed in an air-bath thermostating equipment (homemade) in which the temperature was monitored and controlled by a model WMZK-01 Temperature Controller (Medical Instrument Co., Shanghai). The platinum electrodes were placed parallel in a detection cell (its cell constant was 1.04 cm 1). Not being in contact with the
OSCILLATOR CIRCUITRY
-
-
FREQUENCY COUNTER
F [
~|
COMPUTER
PLOTTER
SBN II RESONRTOR
DETECTION CELL
Fig. 1. Schematic diagram of the experimental setup. Cpc is a parallel capacitor.
solution, such a device should be electrically connected as close as possible to the pair of parallel platinum electrodes set in a 10 ml detection cell. The solution in the cell was stirred with a magnetic stirrer at a constant stirring rate. The oscillation frequency of the SAW resonator was measured by using a model SC7201 Iwatsu Universal Counter at a resolution of 1 Hz, and data were collected at 6 points min -]. The pH of the working buffer was measured by a model EA940 Orion ion analyzer using a calibrated glass electrode. A personal computer was used for data processing. A d.c. power of 8.0 V was supplied by an adjustable dual track d.c. power supply.
Preparation of urease extract Urease from green soya bean was extracted by a simple procedure (Torrey, 1983). No further purification procedures were needed. At first, green soya bean was finely ground using an electric smasher and was screened to pass a 60mesh sieve, then 5.00 g of the powder was weighed and transferred to a 100 ml Erlenmeyer flask with a stopper. Fifty milliliters of organic extracting solvent were added to this vessel. The mixture was shaken on an oscillating machine for 30 min at room temperature and kept in a refrigerator at 4°C for 4 h. The solution was then centrifuged at 2000 g for 10 min. The urease solution was obtained after phase separation. It was stored in a brown flask and kept in a refrigerator before the experiment. The crude extract can be used directly in the measurements below. The urease concentration in this extract was estimated as 25-0 EU ml-~ by comparing its kinetic response with the commercial urease at 2-0 mg m1-1 urea. Experiments have shown that 437
Dezhong Liu et al.
the activity of urease extract can remain steady for about 4 weeks.
Biosensors & Bioelectronics
G.0 /'4
5.0 Frequency response measurements
\
b
.,4.0 4¢.
The whole setup must be turned on for at least 30 min in order to guarantee stabilization before the experiment. The air bath was thermostated at 25 - 0.2°C. A typical procedure was the following: 0.50 ml urease solution and 9.50 ml working buffer were transferred to the detection cell. The temperature of the detection cell was thermostated at 25.0 --- 0.2°C and the solution was stirred by a magnetic stirrer. Frequency drift was recorded prior to adding urea solution, then the steady frequency was recorded (Fo). After a definite volume of standard urea solution was added with a 50 ~1 syringe, the enzyme-catalyzed reaction was started and the oscillation frequency changed with time. Frequency (F) was recorded with time and the frequency shift (AF) was calculated by subtracting Fo from F. The calibration curve was obtained by measurement of the sensor responses to different amounts of the control urine sample. Only a 5.0/.d human urine sample was needed for urea measurement and the urea concentration was estimated by the calibration method.
RESULTS AND DISCUSSION
C
.c 3.0 ~2.0 cO
:
d
1.0
~ 0.0 I
I
1.o 21o 3.0 Time / min
,
I
5.0
Fig. 2. Frequency response of the SAW urea sensor for different concentrations of urease from green soya bean in the reaction system: (a) 1.75; (b) 1.25; (c) 0.75; (d) 0.25 EU m1-1. 0.10 mg m1-1 urea was used in this procedure. 7,0-
~ S.O
c
5.0
cl
~_ 4,0 c-
3,0
~.~ ~.~ I
h
I
,
I
010 1,0 2.0 3.0 4.0 5,0 G,~ Typical kinetic responses for different amounts of urease Typical responses of the SAW urea sensor to standard urea under the condition of different concentrations of urease are shown in Fig. 2. The substrate urea concentration was 0.10 mg m1-1 in this experiment. It can be seen that frequency response increases with the increase of urease concentration, i.e. the reaction rate increases with increasing urease content. The response curve of the SAW urea sensor is analogous to normal enzymatic reaction (Skoog et al., 1988). Selection of extracting solvent Figure 3 has shown the frequency response for the following four extracting solvents: 20% glycerol, 30% ethanol, 32% acetone (all in volume ratio) and water. It can be seen that 438
Time
(min)
Fig. 3. Frequency responses for different kinds of extracting solvent used in the preparation of urease from green soya bean: (a) 20% glycerol; (b) 30% ethanol; (c) 32% acetone; (d) water. 0.20mg m1-1 urea and 1.25 EUm1-1 urease were used in this experiment.
three kinds of organic solvents possess almost the same extracting ability. There is a lower urease activity for water as the extracting solvent than for the other solvents. Twenty percent glycerol was the best extract-solvent and was selected because of its protective effect on urease activity. Effects of pH and temperature Enzymes are very sensitive to changes of pH and function best over a very limited range with a
Biosensors & Bioelectronics
A new urea sensor
a considerable effect on the frequency response (urease activity). Within the range of 34--38°C, the temperature influence can be neglected and the sensor system yields the greatest sensitivity. A temperature of 25°C was selected in our experiment by considering the measurement conditions.
2, 6am ¢,J
¢ > ~ 40 Kinetic
% --
parameters
20
r-t
,
o ,0
I
~
I
6.g
,
?.0
I
,
l°J
8.0
£.0
10.0
pH
Fig. 4. Effect of p H on urease activity. 1.25 EU ml -~ urease extracted from green soya bean and 50 Ixg m1-1 urea were selected in this measurement.
definite pH optimum. The effects of pH are due to changes in the ionic state of the amino acid residues of the enzyme, influencing the binding of the substrate with enzyme and affecting the rate of reaction. Over a narrow pH range, these effects will be reversible. Variations in the pH and temperature of the buffer solution also produce ion strength variations. Calculated amounts of NaCI solution were added to the buffer in such a way that, for each experimental point, a constant value of ionic strength was maintained. Figure 4 shows the effect of the pH of the glycine buffer (1-0 mM) on the SAW urea sensor response over the pH ranges 5.5-9.3. It can be seen that the sensor gives the maximum sensitivity at pH 7.0. Temperature greatly affects the activity of the enzyme. Figure 5 shows that the temperature has
Kinetic parameters of urease to urea depend not only upon its source, reaction pH and temperature, but also upon the type and concentration of buffer. For jack bean urease, the value of the Michaelis constant Km ranges between 1 and 5 mM, according to the experimental conditions (Lynn & Yankwick, 1964; Lynn, 1967). In general, the amino buffer (e.g. glycine) results in a greater enzyme activity than does the simple inorganic buffer such as phosphate or carbonate. In this experiment, 1.0 mM glycine buffer (pH 7.0) and 25.0°C were selected. The Km and maximum rate for the urease from green soya bean were estimated as 2.14 mM and 27.18 kHz min -1 separately, by using Lineweaver-Burk analysis corresponding to substrate concentrations ranging from 1.0 to 20 tzg ml -~. Calibration
Figure 6 depicts typical responses of the SAW urea sensor to standard urea solution in different concentrations. There is a linear correlation
N "r
.,. t00
curves and recovery
\ w
80
7.0
7.g
6.0-
~.0 ~"
5.0
5.8 --
4.0
4.0
3.0
3.~
20 •
2.0
"3
c"
.~
60
I.I
~>
40
--
20
r-
g, 1.0
I N \
[.0 3
t.~
rv,
I
0
tO
,
I
~
l
,
I
~
i
20 30 40 50 T e m p e r a t u r e / °C
0
,
GO
Fig. 5. Effect o f temperature on urease activity. The experimental conditions were the same as in Fig. 4.
10
20 CUrea]
30
40
50
(~g/mL)
Fig. 6. Calibration curves for determination of urea. 1.25 E U m1-1 urease from green soya bean was used in the assay of urea.
439
Dezhong Liu et al.
Biosensors & Bioelectronics
AF = 233.7 [Urea] + 10, n = 8, r = 0.9982
Effect of activators and inhibitors
(4) where the urea concentration is expressed in /xg m1-1 and AF in Hz. The detection limit of the SAW urea sensor as 1.0/~g m1-1 (1.7 × 10 -5 mol 1-1). As derived by Michaelis and Menten (see Palme, 1981) the initial rate (Vo) is given by k[E][Urea] Vo - [Urea] + Km
(5)
where k is the rate constant relating to product formation, [E] is the concentration of urease, and Km is the Michaelis constant. Hence, it is evident that Vo is directly proportional to the content of the enzyme when [Urea] ~ Km and at constant concentration of substrate, so the urea concentration can be detected by using the kinetic method. A correlation of the initial rate vs. urea concentration was presented: Vo = 181.7 [Urea] + 19,
n = 8, r = 0.9990
(6) where the urea concentration is expressed in /zg ml -x and Vo in Hz min -1. The linearity ranges up to 17.5/zg m1-1. The kinetic method can decrease the measurement time but it has a lower sensitivity and a narrower linearity than the fixedtime method by comparing Eqs.(4) and (6). So the fixed-time method was employed for the determination of urea concentration. Recovery experiments have been carried out with normal urine spiked with known urea concentrations. The recovery ranged from 95 to 105% (Table 1).
TABLE 1 Analytical recovery*. Urea (l~g rn1-1) Added
Found
1.00 2.50 5.00 10.0 15.0 20-0
0.95 2.61 4.91 9-62 15.4 19.6
Recovery (%)
95-0 104.4 98.2 96-2 102.3 98.3
Mean (%)
RSD (%)
99.1
3.70
*For experimental conditions see legend to Fig. 6. All results were calculated by the calibration equation. 440
Effects of interferents on frequency response of the SAW urea sensor are shown in Table 2. Results have shown that an amount of activator such as the amino acid glycine can increase the rate of urease reaction in the test solution; there were no observable influences on rate of urease reaction for sodium chloride, glucose, starch, human serumalbumin. Inhibitors such as heavy metal ions (Pb 2÷, Hg 2÷, Ag ÷) decrease the reaction rate and Hg 2÷ and Ag ÷ seriously inhibited urease reaction. Figure 7 shows the frequency-shift response in the presence of different concentrations of inhibitor Hg z+. Results show that the urease reaction rate decreases with increasing content of Hg 2÷ in the test solution, and Hg 2÷ is an effective inhibitor for urease. This property may be used as a method for the determination of mercury(II). Inhibitor-like thiourea structurally resembles the urea and may be bound by the urease, but cannot be converted to the products. Figure 8 shows the influence of thiourea on the relative initial rate of the reaction (i.e. relative activity (RA) of urease). The plot is linear, ranging from 0 to 6.0 mM thiourea: R A (%) = - 1 0 . 4 [Thiourea] + 98.9,
T A B L E 2 Effects of interferents on frequency response of the SAW urea sensor*. lnterferents
None Glucose Soluble starch Human serumalbumin Sodium chloride Zinc chloride Ferric chloride Ferrous sulfate Lead nitrate Cadmium nitrate Silver nitrate Mercury nitrate Thiourea Glycine
Concentration
4.0 mg m1-1 4.0 mg m1-1 2-0 mg m1-1 1-0 mM 0-10 mM 0-10 mM 0.10 mM 0-10 mM 0.10 mM 0.05 mM 1.00/zM 1.00 mM 1.00 mM
Relative response (%) 100 97 98 97 99 95 94 97 95 94 80 85 94 103
*The concentration of urea was 50/~g m1-1 (0.83 mM). For experimental conditions see Experimental section.
A new urea sensor
Biosensors & Bioelectronics
TABLE 3 Assay of urea content in human urine samples.
8.0
Sex ~ \
S.0
b
'*'4.0
Clinical diagnostic results
Tested Normal value value (Ye et al., 1989)
c
.£:
I 0.0
~
I 1.0
1
[ 2.0
T~me
,
, 3.0
I
I 4.0
Content of urea (mg m1-1)
1
/ min
Fig, 7. Responses of the urea sensor to standard urea solutions (50 I~g m1-1) with inhibitor Hg e÷ in different concentrations: (a) O; (b) 1.0," (c) 2.5; (d) 5.0; (e) 7.5 tzM.
Female Female Male Male Male Male Male Female Female Male Male
Healthy Healthy Healthy Healthy Healthy Healthy Chronic nephritis Chronic nephritis Nephrotic syndrome Chronic nephritis Nephrotic syndrome
12.5 12.9 19.8 16.3 12.4 16.7 9.32 8-95 9.90 9.93 7.36
10-20
•~" 1 0 0
patients with liver or kidney diseases, the urea content in urine is lower than the normal value. The tested results reflect the facts and support the clinical diagnosis. The above-mentioned differences of urea content in urine between tested and normal values for renal disease patients have been explained pathologically (Harrington & Zimmerman, 1982), therefore analysis of urea content in urine can deliver important diagnostic evidence.
> 60
_
O
c[
40
) 20
-
a~
0.0
1.0 2.[3 3.0 CThiourea]
4.0 5.0 ( mM )
6.0
Fig. 8. Relative activity of urease as a function of the concentration of inhibitor thiourea. The experimental conditions were the same as in Fig. 4.
n = 7, r = -0.996
(7)
Determination of urea in human urine
Urea concentrations of 11 urine samples were then determined based on the above calibration equation and 5.0/xl of urine samples was used for all assays. The results are summarized in Table 3. The clinical analysis of urea has shown: (1) for healthy adults, the urea concentrations coincide with the literature range (Ye et al., 1989); (2) for
CONCLUSIONS In conclusion, the proposed SAW urea sensor, combining the sensitivity of the SAW sensor device in a relative high concentration electrolyte and the specificity of urease, is well-adapted for routine urea assay of small urine samples. This method is simple and sensitive and provides a uniquely advantageous specific assay of urea.
ACKNOWLEDGEMENTS This work was financially supported by the National Natural Science Foundation of China and Education Commission Fund of China. 441
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