Acetylcholinesterase sensors based on gold electrodes modified with dendrimer and polyaniline

Analytica Chimica Acta 514 (2004) 79–88

Acetylcholinesterase sensors based on gold electrodes modified with dendrimer and polyaniline A comparative research M. Snejdarkova a , L. Svobodova a , G. Evtugyn b , H. Budnikov b , A. Karyakin c , D.P. Nikolelis d , T. Hianik e,∗ a

e

Institute of Animal Biochemistry and Genetics, Slovak Academy of Sciences, 90028 Ivanka pri Dunaji, Slovak Republic b Department of Chemistry, Kazan State University, 18 Kremlevskaya Street, 420008 Kazan, Russia c Department of Chemical Enzymology, Moscow State University, 119899 Moscow, Russia d Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, Panepistimiopolis-Zografou, 15771 Athens, Greece Department of Biophysics and Chemical Physics, Faculty of Mathematics, Physics and Computer Science, Comenius University, Mlynska dol. F1, 84248 Bratislava, Slovak Republic Received 7 April 2003; received in revised form 18 February 2004; accepted 5 March 2004

Abstract Potentiometric and amperometric enzyme sensors based on modified gold electrodes have been developed and compared in pesticide determination. PAMAM dendrimer (generation G4) stabilized with 1-hexadecanethiol was used for the immobilization of acetylcholinesterase from electric eel and choline oxidase from Alcaligenes species in the assembly of amperometric sensor. Polyaniline-doped with camphorsulfonic acid was used to obtain potentiometric response. Trichlorfon, carbofuran and eserine suppress the biosensor response due to their inhibitory effect. The detection limits of 0.003 and 200 nmol l−1 (trichlorfon), 0.04 and 6 nmol l−1 (carbofuran) and 0.1 and 700 nmol l−1 were obtained for amperometric and potentiometric sensors, respectively. The difference in the biosensor behavior and the high sensitivity of the dendrimer modified sensor to the inhibitors is due to the specific organization of protein layer at charged surface of the modifier macromolecules. © 2004 Elsevier B.V. All rights reserved. Keywords: Dendrimers; Polyaniline; Cholinesterase; Enzyme sensor

1. Introduction The increasing amount of potential harmful pollutants released in the environment calls for the development of fast and sensitive analytical techniques for their monitoring. The cost and complexity of traditional analytical methods, e.g. chromatography, limits their application on regular basis, especially in field. In this respect, biosensors are considered as suitable complementary tools for preliminary screening of toxic species and environmental risk assessment. The application of enzymes in the assembly of biosensors developed for environmental monitoring is commonly based on quantification of their inhibition in the presence of hazardous species. Thus, organophoshorus and carbamic pesticides, heavy metals and detergents exert strong spe∗ Corresponding author. Tel.: +421-2-60295683; fax: +421-2-65426774. E-mail address: [email protected] (T. Hianik).

0003-2670/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2004.03.019

cific inhibition of acetylcholinesterase (AChE) which can be measured either amperometrically or potentiometrically. The sensitive and selective detection of the anticholinesterase pollutants was realized in the enzyme sensors of different assembly (see reviews [1–4]). There are two strategies for amperometric detection of AChE activity, i.e. the use of synthetic substrates (thiocholine ethers) Eq. (1) [5,6], or the implementation of a second enzyme providing consecutive conversion of native substrate (acetylcholine) to electrochemically active products. In the first case, acetylthiocholine is enzymatically converted to thiocholine which can be easily oxidized into appropriate disulfide. AChE

acethylthiocholine + H2 O −−→ thiocholine + acetic acid anodic oxidation

2-thiocholine −−−−−−−−→ disulfide + 2H+ (1)

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In the second approach, choline oxidase (ChO) was suggested for this purpose [7–10]. ChO catalyzes the oxidation of choline formed in AChE hydrolysis of the substrate. This results in release of hydrogen peroxide easily oxidized on electrode. AChE acetylcholine + H2 O −−→ choline + acetic acid ChO

choline + 2O2 + H2 O −→ betaine + 2H2 O2

(2)

H2 O2 → 2H+ + O2 + 2e− The current of hydrogen peroxide oxidation can be used as a measure of AChE activity and hence as a response of the AChE–ChO sensor. The oxidation of hydrogen peroxide requires application of the potential to the working electrode with amplitude between 0.4 and 0.7 V (positive terminal on a working electrode). The application of a third enzyme, horseradish peroxidase (HRP), provides lowering this potential due to the promotion of electron transfer from electrode [11]: HRP

H2 O2 + 2e− + 2H+ −→ 2H2 O

(3)

The potentiometric detection of the AChE activity is based on the measurement of the pH shift in the enzymatic layer on the sensor surface that is related to the release of acetic acid (CH3 COOH) formed in the hydrolysis of acetylcholine (see Eq. (2)). Conventional pH sensitive transducers can be used for this purpose, e.g. glass (see [12–14]) and metal oxide electrodes [15] or pH sensitive field effect transistors [16–18]. Recently the advantages of polyaniline (PAN) as transducing material for the pH measurement were reported [19–22]. Chemically synthesized PAN-doped with camphorsulfonic acid showed reversible potentiometric response of approximately 90 mV per pH unit over the pH range from 3 to 9. Both potentiometric and amperometric AChE sensors have their advantages and limitations in inhibitor determination. Potentiometric response is measured in non-current mode and Faradaic processes do not affect enzymatic reaction as it can be expected in the case of amperometric devices. Potentiometric biosensors are also easier for use in field than amperometric devices. However, their response is sensitive to pH value and buffer capacity of the working media and of the samples tested. This is especially important when the signal is measured in mineralized samples, e.g. underwater, extracts from soil, etc. Amperometric biosensors are considered more sensitive and accurate in the substrate determination than potentiometric sensors due to the linear relationship between the current and concentration of the substrate at relatively low substrate concentration. If the response of an amperometric sensor is limited by the rate of enzymatic reaction, the dependence of the signal on a substrate concentration is described by Hanes Eq. (4), an electrochemical form of the Michaelis–Menten equation: C C + KM (4) = I Imax

Here Imax is a maximum steady-state current referring to the saturation of enzyme layer with a substrate, I is the current measured at the concentration C of a substrate and KM an apparent Michaelis–Menten constant. However, the difference in the behavior of amperometric and potentiometric biosensors seems insignificant if they are used for the quantification of irreversible inhibition. Indeed, the decay of the signal after the contact of biosensor with an inhibitor is assigned by the part of enzyme became inactive and should not depend on the measurement mode. In support of this, many investigations show similar analytical characteristics of the determination of AChE inhibitors obtained with amperometric and potentiometric sensors. In certain cases the potentiometric sensors were more sensitive. For example, the lowest detection limit of Malathion (about 10−10 mol l−1 ) was reported for the potentiometric AChE sensor based on Ir/IrO2 electrode [15] whereas amperometric devices were found less sensitive (detection limits about 10−8 mol l−1 ) [5,6]. Detailed comparison of potentiometric and amperometric sensors designed for inhibitor determination is presented in review [23]. Higher sensitivity of amperometric AChE sensors toward inhibitors commonly reported in [2,3] can be rather related to the conditions of enzyme immobilization. High sensitivity and accuracy of the signal measurements typical for amperometric transducers makes it possible to diminish AChE amount required for immobilization whereas potentiometric sensors require higher enzyme loading. The increase in specific concentration of enzyme leads to the decrease in the sensitivity toward inhibitors with no respect to the transducer used. In comparable conditions of AChE immobilization, similar characteristics of inhibitor determination can be reached both for amperometric and potentiometric detection mode (see [3,4,23] and reference herein). The effect of enzyme immobilization on the inhibition measurement emphasizes the necessity of careful investigation of this procedure for the optimization of the AChE sensors. In this work, two novel methods for surface modification and enzyme immobilization were explored and compared in potentiometric and amperometric determination of irreversible AChE inhibitors. Au electrodes were used both for potentiometric and amperometric measurements of the signal. In potentiometric measurements, the electrodes were modified with PAN deposited from chloroform solution. As was shown previously, high pH-sensitivity of PAN modified electrodes improves the performance of appropriate biosensors in comparison with the use of traditional pH-transducers. Thus, urease sensor with enzyme immobilized on screen-printed electrode modified with PAN has been described with detection limit of 1×10−5 mol l−1 urea and maximum signal of about 120 mV [20]. PAN modified glassy carbon electrode was also successfully used for the immobilization of cholinesterases from various sources [22]. For amperometric detection, AChE and ChO were co-immobilized onto the Au electrode modified with PA-

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MAM dendrimer. This support was recently proposed for the immobilization of enzymes due to its high mechanical stability and variety of functional groups available for immobilization [24,25]. The advantages of multi-arrayed enzyme films were shown first for glucose oxidase linked to the fourth generation of dendrimers (G4). We showed also the applicability of dendrimer support for the development of amperometric AChE sensor [26].

2. Experimental 2.1. Preparation of biosensors and chemicals Gold electrodes of 0.3 mm (potentiometric sensors) and 1.6 mm diameter (Minerale, Poland, amperometric sensors) were used for enzyme immobilization. The working surface of electrodes was first polished with 0.3 ␮m alumina powder. Then the electrodes were immersed in the chloroform and extensively cleaned in an ultrasound bath type sonicator (Tesla) during 5 min. After washing in deionized distilled water, the electrodes were cleaned with a hot piranha solution (a 1:3 mixture of 30% H2 O2 and concentrated H2 SO4 ) during 5 min and finally by repeated cycling the potential in range from 0.4 to 1.4 V versus SCE, scan rate 50 mV s−1 , in 0.2 mol l−1 H2 SO4 until an oxidation peak appeared at approximately 0.9 V and remained unchanged. The electrodes were then washed in deionized water (resistance > 15 M cm, ELIX 5, Millipore, El Paso, USA) and for a short period of time in deoxygenated ethanol. The electrodes were then used for the deposition of PAN or preparation of a dendrimer layer followed by the enzyme immobilization. 2.1.1. Enzyme sensor based on a PAN modified electrode PAN in the emeraldine hydrochloride form was synthesized by low-temperature oxidation with ammonium persulfate in 1 mol l−1 HCl as described in [27]. The PAN powder was washed with 1% aqueous ammonia, dried and ground with camphorsulfonic acid and phenol in the 2:1:1 molar ratio (calculated per one p-phenylene imine unit of the polymer). The mixture obtained was dissolved in chloroform to the final PAN concentration of 0.5% (w/w). Then 1 ␮l of the solution was added onto the electrode surface and allowed to dry for 30 min at room temperature. The surface concentration of the PAN corresponded to the maximum pH-sensitivity and stability of the response in accordance with previous investigation [22]. Thick-film epoxygraphite electrodes (IVA Ltd., Ekaterinburg, Russia) were modified with PAN in the same way. AChE (electric eel, EC 3.1.1.7., Sigma, St. Louis, MO, USA, 1170 units mg−1 , solid) was immobilized onto the PAN layer by cross-linking with glutaraldehyde as follows. Bovine serum albumin (BSA, Sigma, 6.5 mg in 1 ml of 0.05 mol l−1 phosphate buffer solution, pH 7.4) and AChE (2 mg in 200 ␮l of the same buffer solution) were mixed

81

to obtain the enzyme concentration required. Then 1 ␮l of the mixture was added onto the surface of electrode and left for 15 min to form a wet film. The electrode was then treated with glutaraldehyde vapors (1% v/v solution) in the water pump vacuum during 2 min. Then the enzyme sensor was dried at room temperature and treated with 0.1 mol l−1 glycine during 10 min to saturate free aldehyde groups. Acetylcholine iodide or chloride (ACh, Sigma) were used as the AChE substrates. No difference in their behavior was observed in all potentiometric experiments. Signal measurements were carried out in 2 mmol l−1 phosphate buffer solution containing 0.1 mol l−1 NaCl, pH 7.8, at room temperature (20 ◦ C). 2.1.2. Enzyme sensor based on the Au electrode modified with dendrimer G4 A self-assembled monolayer containing PAMAM dendrimer (generation G4, Aldrich, San Francisco, CA, USA) was formed on the gold surface as follows: clean Au electrode was immersed into the mixture of 1 mmol l−1 1-hexadecanethiol (Aldrich) and 0.02 mmol l−1 G4 in ethanol for 22 h at room temperature, then rinsed several times by ethanol and deionized water and wetted with the solution of the AChE and ChO from Alcaligenes species (EC 1.1.3.17., 12 units mg−1 solid, Sigma). In accordance with preliminary experiments [26], the enzyme ratio AChE:ChO = 0.45:1 w/w was used in all the experiments. After drying at room temperature, the electrode was wetted with 1 mg ml−1 BSA solution and then with glutaraldehyde vapors as described earlier. To establish the effect of dendrimers, AChE and ChO were also immobilized directly onto the surface of Au electrode in BSA-glutaraldehyde matrix as follows: the enzymes were dissolved in phosphate buffer solution in the ratio of 0.45:1 w/w, and then 2 ␮l of the mixture were added onto the electrode surface and after drying wetted consecutively with 2 ␮l of 1.0 mg ml−1 BSA solution and 2 ␮l of 1% glutaraldehyde solution. The total content of proteins on the electrode corresponded to 3 U cm−2 of AChE, 34 U cm−2 of ChO and about 1.5 mg cm−2 of BSA. After drying, the electrodes were washed with buffer solution and deionized water. The bi-enzyme sensors were kept in dry conditions at 4 ◦ C. The acetylcholine chloride (Sigma) was used as enzyme substrate in all amperometric measurements. The experiments were carried out in 0.1 mol l−1 phosphate buffer solution, pH 7.5, at room temperature (20 ◦ C). 2.2. Electrochemical measurements of biosensor signal In potentiometric measurements, the shift of the potential before and after the substrate/inhibitor injection was measured with precision digital pH meter Ecotest-001 (Econix, Moscow, Russia) versus Ag/AgCl reference electrode. The steady-state signal was reached in 5 min after the substrate addition and used as the sensor signal for the determination

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of the substrate and for the calculation of degree of inhibition. In amperometric measurements, cyclic voltammograms were recorded in dc mode. Bi-enzyme AChE–ChO sensor and clean Au electrode were used as working and auxiliary electrodes and SCE as a reference electrode [28]. The programmable electrometer Keithley 6512 (Keithley, USA) connected with IBM computer through KPC-488.2AT Hi Speed IEEE-Interface Board was used for recording I–E curves. The peak current at +670 mV was measured as a biosensor signal. All the measurements were performed in the Faraday cage at 20 ◦ C with stirring. 2.3. Inhibition measurement and signal regeneration Three typical anticholinesterase compounds, i.e. trichlorfon, carbofuran and eserine (Aldrich) were used in inhibition measurements:

the enzymatic layer and immersed into buffered inhibitor solution. After 10 min incubation, the enzyme sensor was washed again and the response toward ACh was measured as described above. The degree of inhibition, percentage, was calculated as a relative decay of the biosensor response after the contact with an inhibitor. As was previously shown [30], the initial response of cholinesterase sensor can be easily restored after its contact with relatively low concentration of a pesticide (below 1 ppm) by washing. The reactivation was performed with 0.1 mol l−1 phosphate buffer solution containing 2 mmol l−1 ACh for 1 h after each inhibition and overnight at the end of the day. Phosphorylated AChE formed in the incubation with trichlorfon was reactivated with 1 ␮mol l−1 2-pyridine aldoxime methiodide (Sigma). In the above conditions, the amperometric AChE sensors stored at 4 ◦ C at dry conditions showed stable and repro-

(5) Trichlorfon can be converted into more toxic dimethyl-2,2dichlorovinylphosphate (DDVP, dichlorvos) in weakly basic media. For this purpose, the stock solution of trichlorfon was heated to 90 ◦ C and then sharply cooled in the ice bath before the inhibition measurement.

ducible response towards the substrate and inhibitors during 3 weeks. Potentiometric AChE sensors were stored at 4 ◦ C in working buffer solution during at least 2 weeks because of the sharp decay of their activity in dry conditions.

3. Results and discussion (6) This procedure increases the inhibitory effect of trichlorfon by several times and improves the reproducibility of the response after the incubation stage. The DDVP solution obtained in heating/cooling cycle is unstable and should be used in 3–4 h. Carbofuran and eserine belong to the carbamates, which are of great interest and significance due to their broad spectrum of biological activity. The carbamates are increasingly used in agriculture and industry as pesticides, medicines, antiseptics, etc. [3,29]. They were used for inhibition measurements without special pretreatment. The inhibition was performed by the incubation method. The enzyme sensor was first equalized in working buffer solution to reach steady-state background signal. After that, the ACh was added to its final concentration of 0.94 mmol l−1 in amperometric measurements and 1.5 mmol l−1 in potentiometric measurements. Then the novel level of the steady-state current or potential was measured and the difference with the background level was calculated as a measure of the initial AChE activity. After that, the enzyme sensor was washed to remove the substrate from

3.1. Operational characteristics of the AChE sensors Experimental conditions of signal measurement were first optimized to establish the operational characteristics of the response toward ACh. The responses of AChE potentiometric sensor based on PAN and amperometric sensors based on dendrimer layered electrodes to repeating addition of the ACh are presented in Fig. 1. Although the specific activity of the enzyme on the electrode surface differs by several times for various sensors, the average response time was found to be similar, i.e. 1.5 ± 0.4 min for amperometric AChE–ChO sensor and 1.1 ± 0.1 min for potentiometric AChE sensor. The response time was determined as a time necessary for reaching 95% of the steady-state signal. The variation of the enzyme activity at the constant amounts of proteins per electrode attained by BSA does not lead to significant changes in this characteristic. Probably this can be considered as an evidence of proximity of diffusion conditions in dendrimer and protein matrix. Contrary to that, the use of more hydrophobic lipid layer leads to slowing down the changes in signal. The response time of 6 min was reported for amperometric sensor

M. Snejdarkova et al. / Analytica Chimica Acta 514 (2004) 79–88

6 (a)

E, mV

with similar amounts of AChE immobilized to lipid layer by avidin–biotin binding [7]. As was shown for another enzyme, i.e. glucose oxidase, the enzyme activity decreased when enzyme molecules were placed at the air–water interface [31]. In this case the contact of enzyme with non-polar environment (air) resulted in changes in macromolecule conformation that caused in decrease of enzyme activity.

7

120

5

90

4 60

3 2

1 30

0 0

2

4

6

8

10

12

t, min 1.4

I, nA

7

(b)

8

6

1.3

5 4 1.2

2

3

1 1.1

1.0 0

20

40

60

83

80

100

t, min Fig. 1. The response of enzyme sensors towards acetylcholine (arrows correspond to the substrate injection) (a) potentiometric sensor based on Au electrode modified with PAN: 1–0.05, 2–0.15, 3–0.25, 4–0.50, 5–0.75, 6–1.0, 7–1.5 mmol l−1 ; (b) amperometric sensor based on Au electrode modified with dendrimer layer: 1–0.05, 2–0.1, 3–0.15, 4–0.25, 5–0.4, 6–0.57, 7–0.7, 8–0.94 mmol l−1 .

3.1.1. Potentiometric AChE sensor based on Au electrode modified with PAN The dependence of the response of AChE sensor based on PAN modified Au electrode on the amount of the enzyme and the ACh concentration is presented in Fig. 2. The increase in the enzyme quantities used in the immobilization increases the biosensor signal until its saturation at the AChE concentration of 0.03 mg ml−1 . The optimal amount of AChE (approximately 3 U per electrode) corresponds well to the results obtained previously with similar sensors based on glassy carbon electrodes covered with PAN [22]. The use of gold as a transducing material instead of glassy carbon decreases the maximum signal and response time by 10–15%. All the characteristics of the determination of AChE with various potentiometric biosensors are summarized in Table 1. The detection limit refers to the substrate concentration resulting in the shift of the potential which equals the triple standard deviation in the measurement of background potential, i.e. 6 mV. The concentration range refers to the linear part of calibration curve in the plots of E, mV, versus log[ACh, mol l−1 ]. The saturation of the response with ACh concentration for potentiometric sensor based on PAN (Fig. 2) takes place at higher concentrations than those obtained with graphite based potentiometric AChE sensors and ampero-

-1

[ACh], mo l l 1E-4

1E-3

120

150

90

120

60

90

30

60

0 0

4

8

12

16

E, m V

E, mV

1E-5 180

20

AChE activity, U per electrode Fig. 2. The response of potentiometric AChE sensor based on PAN modified electrode on ACh concentration (3 U of enzyme on electrode) and on AChE amounts (1.5 mmol l−1 ACh). The results represent mean ± S.D. calculated from six independent experiments.

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Table 1 Comparative characteristics of ACh determination with potentiometric AChE sensors based on different transducers modified with PAN Electrode material

Gold Glassy carbon [22] Epoxy graphite

Concentration range (mol l−1 ) 10−4

10−3

2× to 1.3 × 2 × 10−4 to 2 × 10−3 1 × 10−4 to 2 × 10−3

Detection limit (mol l−1 ) 10−5

4× 1 × 10−4 5 × 10−5

metric AChE–ChO sensors described below. The Michaelis– Menten constant (KM ) calculated from the calibration curve was found to be equal to that obtained for free enzyme in the same measurement conditions (KM = 0.35 ± 0.07 mmol l−1 ). The changes in the characteristics of the AChE sensors observed on various electrodes modified with PAN can be explained by denser packing of proteins on gold in comparison with carbon materials. On one hand, this diminishes the rate of the substrate transfer to AChE and the response of the enzyme sensor. On the other hand, dense surface packing improves the operational characteristics of biosensor, i.e. shelf life and the accuracy of the response measurement. 3.1.2. Amperometric AChE sensor based on Au electrode modified with dendrimers G4 As mentioned above, the use of the ACh as native enzyme substrate calls for the introduction of the second enzyme, i.e. ChO, in the assembly of the AChE sensor. The pH optimum of these enzymes differs from each other. It corresponds to pH 7.0–8.0 for ChO [32] and 8.0–9.0 for AChE [33]. Thus, the influence of the pH on the response of AChE–ChO sensor has been first investigated. We studied the response of the sensor composed of AChE–ChO and that contained ChO alone. In both cases the enzymes were immobilized on a dendrimer layer. For ChO sensor, 1 U ml−1 of AChE was added to the working solution to provide hydrolysis of ACh. The appropriate responses toward 2.5 mmol l−1 of ACh are presented as pH function on Fig. 3. The maximum 7.0

7.5

8.0

8.5

9.0

I, n A

1.0 12 0.8

I, µA

6.5

Sensitivity (mV per decade)

Response time (min)

120 ± 4 95 ± 5 95 ± 5

1.0–1.2 1.5–2.0 2.5–5.0

Life-time (days) Dry conditions

Buffer solution

<15 <20 <20

5–6 7–8 <10

signal of bi-enzyme sensor was reached at pH 7.5 and that for ChO sensor at pH 8. The current referred to the hydrogen peroxide oxidation in accordance with scheme (2) is proportional to the ACh concentration in the range from 5 × 10−5 to 5 × 10−4 mol l−1 in accordance with Eq. (7) (average values for six measurements). I(nA) = 0.488 ± 0.007 (ACh, mmol l−1 ) + 0.005 ± 0.006,

R = 0.997

(7)

The saturation concentration of ACh is similar to that obtained with potentiometric sensor (1 mmol l−1 ) and exceeds that reported for biosensor with AChE immobilized in a poly(vinyl alcohol) cryogel (100 ␮mol l−1 ) [29]. This proves the proximity of mass transfer conditions for both sensors. The apparent value of Michaelis–Menten constant KM = 0.22 ± 0.06 mmol l−1 calculated in accordance with Hanes equation (4) is about 1.6 times lower than that obtained with free enzyme and potentiometric AChE sensor (see Section 3.1.1 and [34]). Recently, it was shown that dendrimers G4 decrease the KM value of membrane bonded AChE of red blood cells. In the presence of 150 ␮mol l−1 of G4 the KM value was found to be a half of that obtained for native enzyme in reference experiments [35]. This effect can be connected with conformational changes of proteins in the presence of dendrimers [36]. Due to the protonation of the amino groups, the surface of dendrimers should be positively charged at experimental conditions used. Contrary to that, PAN compensates for the surface charge due to the protonation of the polymer so that the kinetic characteristics of the enzymatic reaction are closer to those observed in homogeneous solution with free enzyme. 3.2. Pesticide determination: a comparison

9 0.6 6

0.4

3

0.2

0.0

0 6.5

7.0

7.5

8.0

8.5

9.0

pH Fig. 3. The pH dependence of the response of amperometric AChE–ChO sensor (䊉) and of free AChE measured with ChO sensor (䊊) (0.25 mmol l−1 of ACh, +670 mV vs. SCE).

The inhibition measurements were performed in conditions corresponded to the saturation of the enzymatic layer with the substrate, i.e. at the ACh concentration of 1.5 mmol l−1 for potentiometric sensors and 0.94 mmol l−1 for amperometric AChE sensors. In saturation conditions, the shift in the biosensor response after the contact with the pesticide solution is related only to the enzyme–inhibitor interactions and does not depend on the mass transfer of the substrate/inhibitor from the bulk solution to the enzyme layer. This makes it possible to reach the maximum sensitivity of the pesticide determination. If the concentration of the

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85

Table 2 The determination of pesticides with AChE sensors based on modified gold electrodes Inhibitor

a

B

R

Detection limit (nmol l−1 )

Concentration range (␮mol l−1 )

Potentiometric sensor, inhibition (%) = a + b (log[inhibitor], mol l−1 ); immobilization of AchE in BSA-glutraraldehyde matrix adjacent to PAN Trichlorfon 205 ± 15 32 ± 3 0.977 100 0.2–25 Carbofuran 435 ± 15 54 ± 3 0.991 6 0.01–0.2 Eserinea 790 ± 104 130 ± 20 0.995 700 1.0–2.5 Amperometric sensor, inhibition (%) = a + b ([inhibitor], nmol l−1 ); immobilization of AChE and ChO on dendrimer G4 support Trichlorfon −2.2 ± 9.9 3554 ± 731 0.960 0.003 0.005–0.020 Carbofuran −68 ± 18 1673 ± 234 0.981 0.04 0.05–0.09 Eserinea 1.4 ± 1.2 24 ± 1 0.997 0.1 0.2–2.0 Immobilization of AChE and ChO Trichlorfon −8.5 ± 4.5 Carbofuran −0.5 ± 0.6 Eserinea −15 ± 1

25–70 1.0–20 100–550

substrate chosen is lower, the decay in the enzyme activity due to the inhibition is partially compensated by the enzyme active sites which were free in the response measurement prior to the incubation in an inhibitor solution. This effect of substrate concentration is often observed in inhibition measurement with immobilized enzymes [2,12,15]. The calibration curves were linear in the plots of the relative decay of response (degree of inhibition) against log[inhibitor] for potentiometric sensors and against the inhibitor concentration for amperometric sensors. In the latter case the linear calibration provides higher accuracy of the inhibitor measurement and can be considered an evidence of the absence of diffusion limitations in the substrate/inhibitor transfer from bulk solution to the enzymatic layer. This is probably due to the conformation lability of AChE provided by its immobilization on a rather branchy dendrimer support. The characteristics of inhibitor determination are presented in Table 2. The detection limit of an inhibitor corresponded to its concentration caused the relative decay in the signal equal to the triplicate standard error of the signal toward the substrate, i.e. 9% for potentiometric devices and 6% for amperometric sensors. It should be mentioned that in most experiments the decay of the response after incubation stage was less than 100%, i.e. residual enzyme activity was observed. Thus, in trichlorfon measurements the inhibition degree started diminishing if incubation exceeded 30 min (Fig. 4). The inhibitory effect of carbofuran also increased only in first 30 min of incubation and then becomes approximately constant. Every 5 min of inhibition in carbofuran solution the range from 10 to 30 min increase the degree of inhibition by about 5%. The appropriate detection limits obtained with the potentiometric AChE sensor were found to be 1×10−8 and 6×10−9 mol l−1 of carbofuran at 10 and 15 min incubation, respectively. Concerning the eserine determination, a reproducible decrease of the response was observed only for rather small inhibitor concentrations when the degree of inhibition did not exceed 50–60% (Fig. 5). Probably, this was due to the spontaneous reactivation of the carbamylated AChE during the incuba-

Inhibit ion, %

Without incubation stage.

60

40

20

0

15

30

45

Incubation time, min Fig. 4. The effect of incubation time on the inhibitory effect of 5 ␮mol l−1 of trichlorfon. Potentiometric AChE sensor.

80

Inhibit ion, %

a

in BSA-glutraraldehyde matrix 1.33 ± 0.20 0.982 10 4.50 ± 0.55 0.976 0.2 0.20 ± 0.03 0.965 10

60

40

20

-6.0

-5.8

-5.6

-5.4

-5.2

log[Eserine], mol l

-1

Fig. 5. The determination of eserine with potentiometric AChE sensor. The inhibitor and AChE (0.14 mmol l−1 ) are simultaneously introduced into the working solution (incubation 0 min).

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tion combined with the effect of charged groups of eserine on the allosteric center of enzyme. Similar factors control the AChE inhibition by the excess of the substrate as well as the sensitivity of AChE activity to the metal ions [37]. The protonation of nitrogen atom in eserine molecule results in the appearance of a cationic center able to bind reversibly the AChE active site. The complex formed makes the enzyme inaccessible for attack of irreversible inhibitor. However, the addition of ACh leads to the fast dissociation of the complex and hence to the partial recovery of enzyme activity. This ‘protecting’ effect of cationic substances and reversible inhibitors was described in the investigations of irreversible AChE inhibition [1,3,38] and proposed for prevention of acute poisoning with anticholinesterase agents. The interference of eserine inhibition is much more pronounced for potentiometric sensors. Probably the dendrimer used in amperometric biosensors compensates for the effect of protonated eserine molecules on the AChE due to its own charge. Weakly basic media necessary for the potentiometric measurement of the pH shift, i.e. sensor response, promotes both the reactivation of carbamylated AChE and protonation of eserine. As a result, the preliminary incubation of the potentiometric AChE sensor in eserine solution results in irreproducible changes of the response and the quantification of inhibition can be performed only if the inhibitor and substrate are added at one and the same time, without pre-incubation stage. This decreases the sensitivity of eserine determination in comparison with organophosphates. While the detection limits obtained with PAN modified potentiometric sensor are similar to those reported for other cholinesterase sensors, the amperometric sensors with AChE and ChO co-immobilized onto the dendrimer layer are clearly of advantage. Thus, a detection limit of 2.2 ppb (11 nmol l−1 ) was reported for carbofuran determination, with AChE introduced into graphite–epoxy composite (amperometric detection [39]) and 0.05 ppb (0.24 nmol l−1 ) for AChE and ChO immobilized in the column for the pre-concentration of inhibitor in flow system [40]. The common detection limit of trichlorfon is about 10−7 to 10−8 mol l−1 [1–3,25] with the record low level of 0.07 nmol l−1 obtained in non-stationary conditions of FIA [41]. Previously we obtained the detection limit of eserine to be equal to 95 ppb (4 × 10−7 mol l−1 ), with AChE immobilized on supported bilayer lipid membranes (sBLM) [29]. The immobilization of AChE enzyme on graphite support provides the detection of 0.1 ppb of trichlorfon [25]. The high sensitivity of the amperometric AChE–ChO sensor developed is undoubtedly connected with the peculiarities of dendrimer layer. Due to the ellipsoidal shape of the dendrimers, the surface accessible for enzyme binding is larger than that on the smooth electrode. This provides necessary accessibility of the enzyme active site both to the inhibitor and substrate molecules. Dendrimer matrix effectively stabilizes extremely low concentrations of the AChE at the surface layer. To clarify the role of dendrimer as immobilization matrix in the improvement of the sensitiv-

ity toward AChE inhibitors, we have developed a similar AChE–ChO sensor with the enzymes involved in BSA matrix (see Section 2). The ratio of the enzymes was chosen the same as that in bi-enzyme amperometric sensors. However, the microenvironment of AChE was similar to that in potentiometric devices. Therefore, this makes it possible to establish the role of immobilization conditions and to compare the sensitivity of potentiometric and amperometric sensors in similar working conditions. As it is seen from Table 2, the biosensor based on dendrimer is most sensitive toward inhibitors whereas the sensitivity of potentiometric sensor is the lowest one. The higher sensitivity of amperometric bi-enzyme sensor can be particularly related to the ChO inhibition, either directly or via the changes in the conditions of choline conversion (pH shift, electrostatic effects, etc.). In special experiments, we compared the conditions of enzyme stabilization in dendrimer and BSA matrix. When AChE and ChO were immobilized in BSA matrix instead of dendrimer, minimum amount of AChE providing stable and reproducible signal were found to be about 100 times higher. The concentration of AChE also substantially influences the effect of inhibitors. This is shown in Fig. 6, where inhibition effect of trichlorfon as a function of AChE concentration is presented. The influence of surface concentration of AChE on inhibition measured is shown in Fig. 6 for trichlorfon measurement as an example. Amperometric AChE–ChO sensor with enzymes implemented in the BSA matrix was used for the estimation of inhibitory effect of 50 nmol l−1 of trichlorfon in 10 min incubation. The enzymes were immobilized together with BSA by cross-binding with glutaraldehyde as described in Section 2 on the surface of Au electrode. The consecutive decrease in the AChE activity results in an increase of degree of inhibition followed by a sharp increase of the standard deviation. The enzyme loading chosen for the other experiments presented in Table 2 (3 U per

Inhibition, %

86

100

80

60

40

0.0 1

0.1

1

AChE activity, U per electrode Fig. 6. The plot of inhibition by trichlorfon the amperometric AChE+CHO sensor as a function of the activity of AChE, i.e. different concentration of AChE molecules. AChE + CHO were immobilized on a BSA matrix. The results represent mean±S.D. calculated from six independent experiments.

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electrode) was considered as a compromise between the sensitivity toward inhibitors and accuracy requirements. Immobilization of AChE and ChO by glutaraldehyde binding in the presence of excessive amounts of BSA did not allow avoiding losses in accuracy of measurement and biosensor lifetime when lower amounts of enzymes were used. Contrary to that, dendrimer as an immobilization matrix made it possible to reach at least 100 times lower surface concentration of the enzymes with the standard deviation of inhibition of about 6–7% and biosensor lifetime of at least 3 weeks. Although the immobilization protocol complicates specifying exact volume of enzyme solution used to form wet protein film on dendrimer layer, the ratio of AChE activity for AChE–ChO sensors described can be calculated from the maximum currents measured at the saturating concentrations of ACh. PAN modified potentiometric AChE sensor require further increase of enzyme concentrations necessary for establishing reproducible and stable response. In accordance to that, the sensitivity of inhibitor determination with potentiometric sensor was lower than that of amperometric devices. The substitution of carbon materials on gold yields a more dense packing of surface layers on a positively charged polymer support. This diminishes the surface effects related to the mass transfer of positively charged substrates and inhibitors (in case of protonated eserine). As a result, the detection limits obtained are considerably lower than those of dendrimer based sensors and closer to the characteristics obtained with free AChE. On the other hand, the potentiometric sensors developed exert a wider linear range of inhibitor concentrations. This can be important for the detection of hazardous levels of environmental pollution and of the content of inhibitors in pesticide formulations. Potentiometric biosensors are simpler in use, especially in the field, and in manufacture. They can also set a priority in the food testing. The potential measurements are less affected by the electrochemically active compounds present in the sample to be tested than current measurements in amperometric devices.

4. Conclusions The comparison of potentiometric and amperometric enzyme sensors made on the same support shows the importance of the transducer modification as promising tool for the improvement of the biosensor performance. PAN and dendrimer layers lead to the difference in the charge of the surface layers and in the features of the enzymes immobilized. This results in the remarkable changes of the sensitivity of pesticide determination. The AChE sensor based on dendrimer layers on gold support revealed record low detection limits of trichlorfon (0.03 nmol l−1 ), carbofuran (0.04 nmol l−1 ) and eserine (0.1 nmol l−1 ) due to the specific organization of enzyme microenvironment on the electrode surface. High mechanic stability, long shelf time, easy

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and inexpensive fabrication and simple use of the enzyme sensors developed make them very attractive for practical applications.

Acknowledgements This work was financially supported by the Commission of the European Union in five Framework program (Project No. QLK3-2000-01311), Slovak–Greece program of collaboration in Science and Technology (Project No. GR/Slov/1/02) and Slovak Grant Agency (1/1015/04 to T.H. and 2/4131/04 to M.S). G.E. and A.K. acknowledge also the financial support of INTAS (Grant 00-273) in the part concerning the development of PAN biosensors.

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