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Urease-based biosensor for mercuric ions determination
A¢'rUATORS B
ELSEVIER
Sensors and Actuators B 42 (1997) 233±237
CHEMICAL
Urease-based biosensor for mercuric ions determination Viatcheslav Volotovsky
Young Jung Nam b, Namsoo Kim b,,
Institute of Molecular Biology and Genetics, NASU, I50 Zabolotny St., Kiev 252143, Ukraine b Korea Food Research Institute, Songnam-si, Kyonggi-do 463-420, Republic of Korea
Received 21 April 1997; receivedin revised form 9 June 1997; accepted 11 July 1997
Abstract Urease immobilized into Nation film on the surface of an ion sensitive field effect transistor (ISFET) has been used for heavy metal salt measurement. Enzyme immobilization into a negatively charged polymer seems to cause an increase in the effect of heavy metal inhibition due to cation accumulation in the polymeric matrix. Fifty percent urease inactivation was observed with 0.2 gM Ag (I), 1.5 gM Hg (II) or 5 ~tM Cu (II). To make the urease-based biosensor sensitive only to mercuric ions, it is proposed to add small amounts (up to I00 gM) of NaI into the sample (to suppress sensitivity to Ag ions) and to rewash the sensor in 100 mM solution of EDTA for 5 min to remove Cu ions. Restoration of enzyme activity after Hg (II) inhibition can be obtained by sensor rewashing in a 300 mM solution of NaI for 5 min. © 1997 Elsevier Science S.A. Keywords: Biosensor; Urease inhibition; Heavy metal ions; Charged polymer
1. Introduction Since biosensors utilize bioelements (enzymes, cells et al.) they can be inhibited by some organic (pesticides, for example) or non-organic (heavy metal ions, cyanides and others) toxicants [1]. Owing to extensive usage of noxious chemicals in the industrial and agricultural sectors, the development of inhibitor biosensors is often connected with problems of environmental pollutant determination. A number of biosensors for heavy metal trace measurement have been previously investigated. Some are based on urease because of its stability, rather high sensitivity to mentioned inhibitors and low cost. As inhibitor selectivity of the enzyme is not as high as substrate specificity [2,3], selective rewashing [4] or enzyme arrays [5] were proposed to improve sensor specificity. Also for further development of urease-based biosensor for heavy metal (HM) ions the determination of increase in sensitivity is extremely important. It has been shown [6] that a properly chosen immobilization method could considerably improve enzyme inhibitor sensitivity compared to that of free enzyme (in solution). Our proposal is to immobilize urease in a * Corresponding author. Tel.: + 82 342 7809131; fax: + 82 342 7099876.
0925-4005/97/$17.00 © 1997 Elsevier Science S.A. All rights reserved. PII SO925-4005(97)OO258-X
negatively charged polymer (Nation) in order to accumulate H M cations in the biomatrix, in such a way as to improve sensor sensitivity to H M ions. To make the probe highly selective, a combination of specific additives and selective rewashing techniques can be used.
2. Experimental 2:I. Materials
Urease (EC 3.5.1.5.) with activity 12 IU mg - I was obtained from Biolar (Lithuania). Nation (product #27470-4) was purchased from Aldrich. All other reagents were obtained from Sigma. 2.2. Enzyme immobilization
The biomembrane was formed using the following method. A drop of solution (about 0.1 gl) containing 10% urease in 5 m M Tris-HNO3, pH 7.0 was deposited onto the sensitive area of an ion sensitive field effect transistor (ISFET) and dried at room temperature for 3 rain. Then 2 ~tl of 0.5% Nation solution in ethanol was added to cover the biomembrane and was dried for 5 min.
234
I': Volotovsky et at./Sensors and Actuators B 42 (1997) 233-237
2.3. Sensor design and measurements n-Channel depletion-mode ISFETs were made on a p-Si wafer with orientation (100) and resistivity 7.5 ohm c m - 1 . Si3N 4 layer provides rather high (about 40 mV per pH) hydrogen ion sensitivity. Sensor chip (dimensions 3 x 10 ram) contains two identical ISFETs, one of them is usually covered by biomembrane and second serves as reference (Fig. 1). Home-made electronic equipment has been used for measurement of pH variations in the biomembrane. Enzymatically catalyzed hydrolysis of urea causes pH changes in the biomembrane that are registered with ISFET. Differential signals from working and reference ISFETs was amplified and recorded. If the sensor is immersed into solution, containing inhibitors, these species suppress enzyme activity that results in a reduction of the biosensor response. Initially sensor response to urea addition was recorded. Then the sensor was immersed into buffer solution containing known concentration of heavy metal salt for a fixed time (5 min as a rule). After washing in buffer solution the sensor response to substratum addition was recorded again. The difference in signals before and after inhibition is proportional to inhibitor concentration and incubation time. The measurements have been carried out at room temperature in a glass cell (1.5 ml) filled with 5 mM Tris-HNO3, pH 7.0. The buffer and sample solutions were intensively stirred.
3. Results and discussion
I .
AMPLIFIER
ER
SFET 1 CONTROLLER /
ISFET 2
Fig. 1. Set-up for inhibition measurements with tSFET-based biosenSOL
Special attention should be payed to control the thickness of the polymeric membrane in the case of Nation-based urease biosensor. To protect the immobilized enzyme from removing into the ambient solution, such a film must be thick enough. On the other hand, HM ions are accumulated in the external layers of polymeric membranes, which (in the case of thick membrane) do not contain enzyme molecules, and the inhibition effect is not amplified but reduced. Thus we optimized the thickness of the polymeric membrane (Fig. 3). The highest sensitivity for HM ions with acceptable stability has been obtained for membranes formed with 2 gl of 0.5% Nation solution in ethanol. 100
3. i. Response of urease immobilized into Nation fihn to H M salt inhibition To determine heavy metal (HM) ions, the measurement conditions were properly optimized. In order to get a high 'signal-noise' ratio, measurements of enzyme activity before and after inhibition were carried out in low concentration (5 mM) Tris buffer. Analysis of the biosensor calibration curve allows us to choose the optimal substratum concentration for enzyme activity measurement. For urease-based biosensors the calibration curve reaches a plateau at 5 mM urea concentration, however, previous investigators [2] propose measuring sensor response with excess urea. Therefore we injected three times higher urea concentration, which corresponds to the beginning of the saturation region-- 15 mM. tt was reported in [3] that HM ions inhibit free urease in the following order: Ag (I)> Hg (II)> Cu (It), and for urease, immobilized into Nation film, the inhibition order corresponds to that of free enzyme (Fig. 2).
2
80
~E "d {ll
60
E 40
20
.1
,2
.3
.6
1
2
3
6
10
Inhibitor concentration, #M
Fig. 2. The calibration curves for H M ion determination by urease biosensor with enzyme immobilized into Nation film: II, Hg (II); e, Ag (I); A, Cu(II). Inhibition time is 5 min.
235
V. Volotovsky et al./Sensors and Actuators B 42 (1997) 2 3 3 - 2 3 7 100
80
60 ._> ~J 09
E ~" 40 W
20
I
1
[
2
3
6
10
20
30
Hg (11), ,uM
Fig. 3. Dependence of biosensor response to Hg (II) on polymeric membrane thickness. Membrane was formed with: O, 2 gl of 0.5% Nation; i , 3 lal of 0.5% Nation; i,, 4 gl of 0.5% Nation.
3.2. Influence of temperature, p H and ionic strength on biosensor sensitivity to H M ions Investigation of temperature influence on the inhibition process is important for several reasons. First, under real measurement conditions the temperature of the sample, containing pollutant, depends on the season and can change within the range 0-30°C. Therefore such a temperature difference can introduce an error into the measured pollutant concentration. Secondly, it was supposed, that increasing the sample temperature would improve inhibition efficiency due to a more intensive interaction of HM ions with the enzyme active site. Experimental results (Fig. 4) have shown a dependence of the inhibition efficiency on sample temperature. Normally the measurements were carried out at room temperature ( + 20°C) and 50% enzyme inhibition under these conditions corresponds to 1.5 laM Hg (II). However a decrease in incubation temperature reduces slightly the inhibition efficiency and at +4°C 50% inhibition can be obtained with 4.5 ~tM Hg (II). In order to investigate the influence of increased temperature on the inhibition process, at first, sensor incubation in warmed buffer solution without any inhibitor was performed. It has been found that biosensor washing in heated (up to +60°C) buffer for 5 min does not suppress enzyme activity. Calibration curves for sensor inhibition with warmed pollutant samples were made. It should be noted, that though inhibition was carried out in HM samples with different temperatures, measurements of enzyme activity (response to urea) was made
at the same conditions (Tris buffer, pH 7.0, at + 20°C). The highest sensitivity to pollutant has been obtained in the case of warmed (up to + 60°C) sample. At this temperature 50% enzyme inhibitions corresponds to 0.7 ~tM Hg (II). However, this slight gain seems to be rather small to regard temperature increase as considerable improvement of enzyme sensitivity to HM ions. As HM ion measurements can be carried out in the samples with considerable salt concentration (sea water, for example), it is necessary to investigate the influence of high ionic strength of the sample on the sensor response. It has been shown [3] that the effect of NaC1 on the inhibition by HM ions was negligible. At the same time high ionic strength of solution can influence some characteristics of the ion-exchange polymer [7] (Nation, in our case). Addition of 100 mM of NaC1 into model inhibitor solution has not led to any notable changes in the sensor sensitivity. Also no variation was recorded in sensor sensitivity to HM ions when the sample pH changed from 6 to 8.
3.3. Improving the biosensor selectivity to mercuric ions Investigators of urease-based HM ion biosensors have recorded poor inhibitor selectivity of enzyme. Calibration curves for different ions are usually overlapping which causes cross-sensitivity [2] and even some synergistic phenomena [3]. Also, mercuric ions are much more dangerous for the human organism than cupric ones, though both inhibit urease being at the same concentration range (1-10 gM). These facts convinced us improve sensor selectivity to one ion. Because of their high toxicity mercuric ions have been chosen. 100
80
>:, 60
C0
N
,,=,
40
20
[
.6
1
2
3
6
10
Hg (11), #M
Fig. 4. Dependence of biosensor response to Hg (II) on the sample temperature: O, +60°C; II, +40°C; A, +20°C; ~', +4°C.
236
V. Volotovsky et al. / Sensors and Actuators B 42 (i997) 233-237 was immersed into a mixture, containing 8 IaM Hg (II), 80 gM Ag (I), 80 laM Cu (II) and 100 gM NaI, after 5 min inhibition and following 5 rain rewashing in 100 mM EDTA, sensor response to urea was about 50% of the original value. This corresponds to enzyme activity after inhibition in solution, containing 8 gM Hg (II) and 100 gM NaI (Fig. 5).
100
80
,~
60
._>
3.4. Sensor stability and reproducibility
CO
E
40 1.U
20
I
L
I
.1
1
10
100
Inhibitor concentration, gM
Fig. 5. Decreasingsensor sensitivityto Ag (I) by NaI addition: o, Ag (I) in buffer without NaI; ,2,, Ag (I) in buffer with I00/aM NaI; i , Hg (II) in buffer without NaI; [], Hg (II) in buffer with 100 gM NaI. To suppress enzyme sensitivity to silver ions it was proposed to add into the sample anions that form insoluble or low soluble salts with silver cations but do not influence the inhibition properties of mercuric ions. The most effective seems to be iodide anions. Even slight addition of NaI into the sample solution sufficiently decreases sensor sensitivity to sitver ions. So, 100 gM concentration of NaI in sample solution protects the enzyme from Ag inhibition by up to 95 gM (Fig. 5). It should be noted that iodide additions cause some decrease in sensor sensitivity to Hg. But a several micromole shift of the calibration curve for mercuric ions seems to be an acceptable price to pay for an almost 1000-fold decrease of silver ion influence. It is not possible to effectively protect enzyme from cupric ion inhibition. In our study, however, it has been shown that enzyme, inhibited with Cu(II) restored its activity after 5 min rewashing in I00 mM EDTA solution. At the same time EDTA did not restore activity of urease, inhibited with Hg (II). Such a selective rewashing makes the biosensor non-sensitive to cupric ions. In order to investigate sensor performance in inhibitor mixture, the following experiment has been carried out. First the sensor response to 15 mM urea addition has been recorded. Then a probe was deposited into the sample solution, containing 80 gM Ag (I), 80 IaM Cu (II) and 100 ~tM NaI for 5 min. After washing in fresh buffer the sensor did not respond to urea addition as enzyme activity was suppressed by cupric ions. After 5 rain rewashing in 100 mM EDTA the response was 100% of the original. When the probe
It has been found that mercuric ions can be removed from the enzyme active center by sensor rewashing in 300 mM NaI solution for 5 min. Though enzyme activity can be restored up to 95-100%, sensor sensitivity to inhibitor is usually changed--increased (Fig. 6). A possible explanation for such a phenomena may be the fact, that reaction of HM ions with non-essential sulfhydryl groups does not lead to enzyme inactivation but to inhibitor consumption [1]. Incubation of the enzyme probe in low concentrated inhibitor solution saturates non-essential groups, so the next portion of inhibitor ions binds with sulfhydryl groups of the active site, and this results in a considerable decrease of enzyme activity. This provides the potential possibility to improve sensor sensitivity by preincubation in a low concentrated inhibitor solution. It has been obtained that 50% urease inhibition can be achieved with 1.5 +_0.5 gM Hg (II) when measurements were carried out with newly prepared sensors in Tris-HNO3 buffer without any admixtures and when the incubation time was 5 rain. Such a variation can be explained by difference in polymeric membrane thickness that cannot be controlled precisely using a manual procedure. Further improvement in membrane deposition technique is still necessary to achieve better sensor reproducibility.
100
0
R
R
R 80
~d
60
©
E LU
40
20 5
Fig. 6. Reproducibilityof biosensor: O, original activity;R, restored activity; 1-6, enzymeactivityafter 5 min inhibition in 1 FM Hg (II).
V. Volotovsky et a l . / Sensors and Actuators B 42 (1997) 2.35-237
It should be noted that newly made biosensors can be stored in air at room temperature for at least 1 month without any loss in sensitivity. Inhibited biomembrane can be removed easily by sensor washing in ethanol solution and an ISFET transducer can be used up to several dozen times.
4. Conclusions ISFET-based biosensor with urease immobilized into a negatively charged polymeric film can be used for the estimation of general water pollution with HM and for selective determination of mercuric ions in aqueous media. As the biosensor detection limit for Hg (II) is about 1 gM (0.2 mg kg-1), a probe can be used for mercury determination in fish products where the tolerated concentration is cited as 0.5 mg kg-1 (USA, Canada) or even 1.0 mg k g - i (Sweden, Denmark, Japan) [8]. For Cu (II) sensor to show a detection limit at 3 laM (0.2 mg kg-1), there must be sufficient precision to determine Cu (II) in food samples as recommended by the National Academy of Sciences (USA) where daily intake is 1.5-3 mg [9]. High sensor selectivity to mercuric ions can be obtained by slight additions (up to 100 laM) of NaI into sample solution (to suppress Ag influence) and by 5 rain sensor rewashing in 100 mM solution of EDTA to remove Cu ions. Though restoration of enzyme activity after Hg inhibition up to 95-100% of original activity has been obtained by 5 rain rewashing in 300 mM NaI solution, sensitivity to inhibitor was not reproducible enough to re-use the same probe many times. Further development of the described biosensor is still necessary. This will include searching for more effective charged polymer for HM ion accumulation and sensor dynamic range extension.
Acknowledgements Financial support from the Korea Science and Engineering Foundation (KOSEF) is gratefully acknowledged.
References [I] J.R. Whitaker, Principles of Enzymology for the Food Sciences,
237
Marcel Dekker, New York, 1972, pp. 255-286. [2] G.A. Zhylyak, S.V. Dzyadevich, Y.I. Korpan, A.P. Soldatkin, A.V. El'skaya, Application of urease conductometric biosensor for heavy-metal ion determination, Sensors and Actuators B 24/25 (1995) 145-148. [3] C. Preininger, O. Wolfbeis, Disposable cuvette test with integrated sensor layer for enzymatic determination of heavy metals, Biosens. Bioelectron. 1i (10) (I996)981-990. [4] C. Tranh-Minh, Immobilized enzyme probes for determining inhibitors, Ion-Selective Electrode Rev. 7 (1985) 41-75. [5] D.C. Cowell, A.A. Dowmann, T. Ashcroft, The detection and identification of metal and organic pollutants in potable water using enzyme assays suitable for sensor development, Biosens. Bioelectron. 10 (6/7) (1995) 509-516. [6] J.C. Gayet, A. Haouz, A. Geloso-Meyer, C. Burstein, Detection of heavy metal salts with biosensors built with an oxygen electrode coupled to various immobilized oxidases and dehydrogenases, Biosens. Bioelectron. 8 (3/4) (1993) 177-183. [7] V. Volotovsky, A.P. Soldatkin, A.A. Shulga, V.K. Rossokhaty, V.I. Strikha, A.V. El'skaya, Glucose sensitive field-effect transistor-based biosensor with additional positively charged membrane. Dynamic range extension and reduction of buffer concentration influence on the sensor response, Anal. Chim. Acta 322 (1996) 77-8i. [8] J.M. Concon, Food Toxicology. Contaminants and Additives, Marcel Dekker, New York, 1988, pp. 1033-1050. [9] F.H. Nielsen, Trace Elements (Nutrition), in: R. Dulbecco (ed.), Encyclopedia of Human Biology, vol. 7, Academic Press, San Diego, I991, pp. 603-613.
Biographies Viatcheslav Volotovsky was born in 1967, graduated from Shevchenko Kiev University in 1993 and received his Ph.D. degree in biotechnology in 1996. He joined the Institute of Molecular Biology and Genetics in 1996 and now holds a research scientist position. His research interests mainly concern enzyme-based biosensors. Young Jung Nam was born in 1940, graduated from Seoul National University in 1963 and received his Ph.D. degree in microbiology in 1986. He joined the Korea Food Research Institute in 1988. His current interests are in biology and health effects of lactic acid bacteria. Namsoo Kim was born in 1955, received his Ph.D. degree in 1992 from Seoul National University. He joined the Korea Food Research Institute in 1988 and now holds a senior research position. Since t993 he has worked on the development of enzyme-based and immuno-biosensors.
ELSEVIER
Sensors and Actuators B 42 (1997) 233±237
CHEMICAL
Urease-based biosensor for mercuric ions determination Viatcheslav Volotovsky
Young Jung Nam b, Namsoo Kim b,,
Institute of Molecular Biology and Genetics, NASU, I50 Zabolotny St., Kiev 252143, Ukraine b Korea Food Research Institute, Songnam-si, Kyonggi-do 463-420, Republic of Korea
Received 21 April 1997; receivedin revised form 9 June 1997; accepted 11 July 1997
Abstract Urease immobilized into Nation film on the surface of an ion sensitive field effect transistor (ISFET) has been used for heavy metal salt measurement. Enzyme immobilization into a negatively charged polymer seems to cause an increase in the effect of heavy metal inhibition due to cation accumulation in the polymeric matrix. Fifty percent urease inactivation was observed with 0.2 gM Ag (I), 1.5 gM Hg (II) or 5 ~tM Cu (II). To make the urease-based biosensor sensitive only to mercuric ions, it is proposed to add small amounts (up to I00 gM) of NaI into the sample (to suppress sensitivity to Ag ions) and to rewash the sensor in 100 mM solution of EDTA for 5 min to remove Cu ions. Restoration of enzyme activity after Hg (II) inhibition can be obtained by sensor rewashing in a 300 mM solution of NaI for 5 min. © 1997 Elsevier Science S.A. Keywords: Biosensor; Urease inhibition; Heavy metal ions; Charged polymer
1. Introduction Since biosensors utilize bioelements (enzymes, cells et al.) they can be inhibited by some organic (pesticides, for example) or non-organic (heavy metal ions, cyanides and others) toxicants [1]. Owing to extensive usage of noxious chemicals in the industrial and agricultural sectors, the development of inhibitor biosensors is often connected with problems of environmental pollutant determination. A number of biosensors for heavy metal trace measurement have been previously investigated. Some are based on urease because of its stability, rather high sensitivity to mentioned inhibitors and low cost. As inhibitor selectivity of the enzyme is not as high as substrate specificity [2,3], selective rewashing [4] or enzyme arrays [5] were proposed to improve sensor specificity. Also for further development of urease-based biosensor for heavy metal (HM) ions the determination of increase in sensitivity is extremely important. It has been shown [6] that a properly chosen immobilization method could considerably improve enzyme inhibitor sensitivity compared to that of free enzyme (in solution). Our proposal is to immobilize urease in a * Corresponding author. Tel.: + 82 342 7809131; fax: + 82 342 7099876.
0925-4005/97/$17.00 © 1997 Elsevier Science S.A. All rights reserved. PII SO925-4005(97)OO258-X
negatively charged polymer (Nation) in order to accumulate H M cations in the biomatrix, in such a way as to improve sensor sensitivity to H M ions. To make the probe highly selective, a combination of specific additives and selective rewashing techniques can be used.
2. Experimental 2:I. Materials
Urease (EC 3.5.1.5.) with activity 12 IU mg - I was obtained from Biolar (Lithuania). Nation (product #27470-4) was purchased from Aldrich. All other reagents were obtained from Sigma. 2.2. Enzyme immobilization
The biomembrane was formed using the following method. A drop of solution (about 0.1 gl) containing 10% urease in 5 m M Tris-HNO3, pH 7.0 was deposited onto the sensitive area of an ion sensitive field effect transistor (ISFET) and dried at room temperature for 3 rain. Then 2 ~tl of 0.5% Nation solution in ethanol was added to cover the biomembrane and was dried for 5 min.
234
I': Volotovsky et at./Sensors and Actuators B 42 (1997) 233-237
2.3. Sensor design and measurements n-Channel depletion-mode ISFETs were made on a p-Si wafer with orientation (100) and resistivity 7.5 ohm c m - 1 . Si3N 4 layer provides rather high (about 40 mV per pH) hydrogen ion sensitivity. Sensor chip (dimensions 3 x 10 ram) contains two identical ISFETs, one of them is usually covered by biomembrane and second serves as reference (Fig. 1). Home-made electronic equipment has been used for measurement of pH variations in the biomembrane. Enzymatically catalyzed hydrolysis of urea causes pH changes in the biomembrane that are registered with ISFET. Differential signals from working and reference ISFETs was amplified and recorded. If the sensor is immersed into solution, containing inhibitors, these species suppress enzyme activity that results in a reduction of the biosensor response. Initially sensor response to urea addition was recorded. Then the sensor was immersed into buffer solution containing known concentration of heavy metal salt for a fixed time (5 min as a rule). After washing in buffer solution the sensor response to substratum addition was recorded again. The difference in signals before and after inhibition is proportional to inhibitor concentration and incubation time. The measurements have been carried out at room temperature in a glass cell (1.5 ml) filled with 5 mM Tris-HNO3, pH 7.0. The buffer and sample solutions were intensively stirred.
3. Results and discussion
I .
AMPLIFIER
ER
SFET 1 CONTROLLER /
ISFET 2
Fig. 1. Set-up for inhibition measurements with tSFET-based biosenSOL
Special attention should be payed to control the thickness of the polymeric membrane in the case of Nation-based urease biosensor. To protect the immobilized enzyme from removing into the ambient solution, such a film must be thick enough. On the other hand, HM ions are accumulated in the external layers of polymeric membranes, which (in the case of thick membrane) do not contain enzyme molecules, and the inhibition effect is not amplified but reduced. Thus we optimized the thickness of the polymeric membrane (Fig. 3). The highest sensitivity for HM ions with acceptable stability has been obtained for membranes formed with 2 gl of 0.5% Nation solution in ethanol. 100
3. i. Response of urease immobilized into Nation fihn to H M salt inhibition To determine heavy metal (HM) ions, the measurement conditions were properly optimized. In order to get a high 'signal-noise' ratio, measurements of enzyme activity before and after inhibition were carried out in low concentration (5 mM) Tris buffer. Analysis of the biosensor calibration curve allows us to choose the optimal substratum concentration for enzyme activity measurement. For urease-based biosensors the calibration curve reaches a plateau at 5 mM urea concentration, however, previous investigators [2] propose measuring sensor response with excess urea. Therefore we injected three times higher urea concentration, which corresponds to the beginning of the saturation region-- 15 mM. tt was reported in [3] that HM ions inhibit free urease in the following order: Ag (I)> Hg (II)> Cu (It), and for urease, immobilized into Nation film, the inhibition order corresponds to that of free enzyme (Fig. 2).
2
80
~E "d {ll
60
E 40
20
.1
,2
.3
.6
1
2
3
6
10
Inhibitor concentration, #M
Fig. 2. The calibration curves for H M ion determination by urease biosensor with enzyme immobilized into Nation film: II, Hg (II); e, Ag (I); A, Cu(II). Inhibition time is 5 min.
235
V. Volotovsky et al./Sensors and Actuators B 42 (1997) 2 3 3 - 2 3 7 100
80
60 ._> ~J 09
E ~" 40 W
20
I
1
[
2
3
6
10
20
30
Hg (11), ,uM
Fig. 3. Dependence of biosensor response to Hg (II) on polymeric membrane thickness. Membrane was formed with: O, 2 gl of 0.5% Nation; i , 3 lal of 0.5% Nation; i,, 4 gl of 0.5% Nation.
3.2. Influence of temperature, p H and ionic strength on biosensor sensitivity to H M ions Investigation of temperature influence on the inhibition process is important for several reasons. First, under real measurement conditions the temperature of the sample, containing pollutant, depends on the season and can change within the range 0-30°C. Therefore such a temperature difference can introduce an error into the measured pollutant concentration. Secondly, it was supposed, that increasing the sample temperature would improve inhibition efficiency due to a more intensive interaction of HM ions with the enzyme active site. Experimental results (Fig. 4) have shown a dependence of the inhibition efficiency on sample temperature. Normally the measurements were carried out at room temperature ( + 20°C) and 50% enzyme inhibition under these conditions corresponds to 1.5 laM Hg (II). However a decrease in incubation temperature reduces slightly the inhibition efficiency and at +4°C 50% inhibition can be obtained with 4.5 ~tM Hg (II). In order to investigate the influence of increased temperature on the inhibition process, at first, sensor incubation in warmed buffer solution without any inhibitor was performed. It has been found that biosensor washing in heated (up to +60°C) buffer for 5 min does not suppress enzyme activity. Calibration curves for sensor inhibition with warmed pollutant samples were made. It should be noted, that though inhibition was carried out in HM samples with different temperatures, measurements of enzyme activity (response to urea) was made
at the same conditions (Tris buffer, pH 7.0, at + 20°C). The highest sensitivity to pollutant has been obtained in the case of warmed (up to + 60°C) sample. At this temperature 50% enzyme inhibitions corresponds to 0.7 ~tM Hg (II). However, this slight gain seems to be rather small to regard temperature increase as considerable improvement of enzyme sensitivity to HM ions. As HM ion measurements can be carried out in the samples with considerable salt concentration (sea water, for example), it is necessary to investigate the influence of high ionic strength of the sample on the sensor response. It has been shown [3] that the effect of NaC1 on the inhibition by HM ions was negligible. At the same time high ionic strength of solution can influence some characteristics of the ion-exchange polymer [7] (Nation, in our case). Addition of 100 mM of NaC1 into model inhibitor solution has not led to any notable changes in the sensor sensitivity. Also no variation was recorded in sensor sensitivity to HM ions when the sample pH changed from 6 to 8.
3.3. Improving the biosensor selectivity to mercuric ions Investigators of urease-based HM ion biosensors have recorded poor inhibitor selectivity of enzyme. Calibration curves for different ions are usually overlapping which causes cross-sensitivity [2] and even some synergistic phenomena [3]. Also, mercuric ions are much more dangerous for the human organism than cupric ones, though both inhibit urease being at the same concentration range (1-10 gM). These facts convinced us improve sensor selectivity to one ion. Because of their high toxicity mercuric ions have been chosen. 100
80
>:, 60
C0
N
,,=,
40
20
[
.6
1
2
3
6
10
Hg (11), #M
Fig. 4. Dependence of biosensor response to Hg (II) on the sample temperature: O, +60°C; II, +40°C; A, +20°C; ~', +4°C.
236
V. Volotovsky et al. / Sensors and Actuators B 42 (i997) 233-237 was immersed into a mixture, containing 8 IaM Hg (II), 80 gM Ag (I), 80 laM Cu (II) and 100 gM NaI, after 5 min inhibition and following 5 rain rewashing in 100 mM EDTA, sensor response to urea was about 50% of the original value. This corresponds to enzyme activity after inhibition in solution, containing 8 gM Hg (II) and 100 gM NaI (Fig. 5).
100
80
,~
60
._>
3.4. Sensor stability and reproducibility
CO
E
40 1.U
20
I
L
I
.1
1
10
100
Inhibitor concentration, gM
Fig. 5. Decreasingsensor sensitivityto Ag (I) by NaI addition: o, Ag (I) in buffer without NaI; ,2,, Ag (I) in buffer with I00/aM NaI; i , Hg (II) in buffer without NaI; [], Hg (II) in buffer with 100 gM NaI. To suppress enzyme sensitivity to silver ions it was proposed to add into the sample anions that form insoluble or low soluble salts with silver cations but do not influence the inhibition properties of mercuric ions. The most effective seems to be iodide anions. Even slight addition of NaI into the sample solution sufficiently decreases sensor sensitivity to sitver ions. So, 100 gM concentration of NaI in sample solution protects the enzyme from Ag inhibition by up to 95 gM (Fig. 5). It should be noted that iodide additions cause some decrease in sensor sensitivity to Hg. But a several micromole shift of the calibration curve for mercuric ions seems to be an acceptable price to pay for an almost 1000-fold decrease of silver ion influence. It is not possible to effectively protect enzyme from cupric ion inhibition. In our study, however, it has been shown that enzyme, inhibited with Cu(II) restored its activity after 5 min rewashing in I00 mM EDTA solution. At the same time EDTA did not restore activity of urease, inhibited with Hg (II). Such a selective rewashing makes the biosensor non-sensitive to cupric ions. In order to investigate sensor performance in inhibitor mixture, the following experiment has been carried out. First the sensor response to 15 mM urea addition has been recorded. Then a probe was deposited into the sample solution, containing 80 gM Ag (I), 80 IaM Cu (II) and 100 ~tM NaI for 5 min. After washing in fresh buffer the sensor did not respond to urea addition as enzyme activity was suppressed by cupric ions. After 5 rain rewashing in 100 mM EDTA the response was 100% of the original. When the probe
It has been found that mercuric ions can be removed from the enzyme active center by sensor rewashing in 300 mM NaI solution for 5 min. Though enzyme activity can be restored up to 95-100%, sensor sensitivity to inhibitor is usually changed--increased (Fig. 6). A possible explanation for such a phenomena may be the fact, that reaction of HM ions with non-essential sulfhydryl groups does not lead to enzyme inactivation but to inhibitor consumption [1]. Incubation of the enzyme probe in low concentrated inhibitor solution saturates non-essential groups, so the next portion of inhibitor ions binds with sulfhydryl groups of the active site, and this results in a considerable decrease of enzyme activity. This provides the potential possibility to improve sensor sensitivity by preincubation in a low concentrated inhibitor solution. It has been obtained that 50% urease inhibition can be achieved with 1.5 +_0.5 gM Hg (II) when measurements were carried out with newly prepared sensors in Tris-HNO3 buffer without any admixtures and when the incubation time was 5 rain. Such a variation can be explained by difference in polymeric membrane thickness that cannot be controlled precisely using a manual procedure. Further improvement in membrane deposition technique is still necessary to achieve better sensor reproducibility.
100
0
R
R
R 80
~d
60
©
E LU
40
20 5
Fig. 6. Reproducibilityof biosensor: O, original activity;R, restored activity; 1-6, enzymeactivityafter 5 min inhibition in 1 FM Hg (II).
V. Volotovsky et a l . / Sensors and Actuators B 42 (1997) 2.35-237
It should be noted that newly made biosensors can be stored in air at room temperature for at least 1 month without any loss in sensitivity. Inhibited biomembrane can be removed easily by sensor washing in ethanol solution and an ISFET transducer can be used up to several dozen times.
4. Conclusions ISFET-based biosensor with urease immobilized into a negatively charged polymeric film can be used for the estimation of general water pollution with HM and for selective determination of mercuric ions in aqueous media. As the biosensor detection limit for Hg (II) is about 1 gM (0.2 mg kg-1), a probe can be used for mercury determination in fish products where the tolerated concentration is cited as 0.5 mg kg-1 (USA, Canada) or even 1.0 mg k g - i (Sweden, Denmark, Japan) [8]. For Cu (II) sensor to show a detection limit at 3 laM (0.2 mg kg-1), there must be sufficient precision to determine Cu (II) in food samples as recommended by the National Academy of Sciences (USA) where daily intake is 1.5-3 mg [9]. High sensor selectivity to mercuric ions can be obtained by slight additions (up to 100 laM) of NaI into sample solution (to suppress Ag influence) and by 5 rain sensor rewashing in 100 mM solution of EDTA to remove Cu ions. Though restoration of enzyme activity after Hg inhibition up to 95-100% of original activity has been obtained by 5 rain rewashing in 300 mM NaI solution, sensitivity to inhibitor was not reproducible enough to re-use the same probe many times. Further development of the described biosensor is still necessary. This will include searching for more effective charged polymer for HM ion accumulation and sensor dynamic range extension.
Acknowledgements Financial support from the Korea Science and Engineering Foundation (KOSEF) is gratefully acknowledged.
References [I] J.R. Whitaker, Principles of Enzymology for the Food Sciences,
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Marcel Dekker, New York, 1972, pp. 255-286. [2] G.A. Zhylyak, S.V. Dzyadevich, Y.I. Korpan, A.P. Soldatkin, A.V. El'skaya, Application of urease conductometric biosensor for heavy-metal ion determination, Sensors and Actuators B 24/25 (1995) 145-148. [3] C. Preininger, O. Wolfbeis, Disposable cuvette test with integrated sensor layer for enzymatic determination of heavy metals, Biosens. Bioelectron. 1i (10) (I996)981-990. [4] C. Tranh-Minh, Immobilized enzyme probes for determining inhibitors, Ion-Selective Electrode Rev. 7 (1985) 41-75. [5] D.C. Cowell, A.A. Dowmann, T. Ashcroft, The detection and identification of metal and organic pollutants in potable water using enzyme assays suitable for sensor development, Biosens. Bioelectron. 10 (6/7) (1995) 509-516. [6] J.C. Gayet, A. Haouz, A. Geloso-Meyer, C. Burstein, Detection of heavy metal salts with biosensors built with an oxygen electrode coupled to various immobilized oxidases and dehydrogenases, Biosens. Bioelectron. 8 (3/4) (1993) 177-183. [7] V. Volotovsky, A.P. Soldatkin, A.A. Shulga, V.K. Rossokhaty, V.I. Strikha, A.V. El'skaya, Glucose sensitive field-effect transistor-based biosensor with additional positively charged membrane. Dynamic range extension and reduction of buffer concentration influence on the sensor response, Anal. Chim. Acta 322 (1996) 77-8i. [8] J.M. Concon, Food Toxicology. Contaminants and Additives, Marcel Dekker, New York, 1988, pp. 1033-1050. [9] F.H. Nielsen, Trace Elements (Nutrition), in: R. Dulbecco (ed.), Encyclopedia of Human Biology, vol. 7, Academic Press, San Diego, I991, pp. 603-613.
Biographies Viatcheslav Volotovsky was born in 1967, graduated from Shevchenko Kiev University in 1993 and received his Ph.D. degree in biotechnology in 1996. He joined the Institute of Molecular Biology and Genetics in 1996 and now holds a research scientist position. His research interests mainly concern enzyme-based biosensors. Young Jung Nam was born in 1940, graduated from Seoul National University in 1963 and received his Ph.D. degree in microbiology in 1986. He joined the Korea Food Research Institute in 1988. His current interests are in biology and health effects of lactic acid bacteria. Namsoo Kim was born in 1955, received his Ph.D. degree in 1992 from Seoul National University. He joined the Korea Food Research Institute in 1988 and now holds a senior research position. Since t993 he has worked on the development of enzyme-based and immuno-biosensors.