Abstract: An bioelectrochemical sensor or biosensor based on the inhibition of the enzyme Alkaline Phosphatase (ALP) has been investigated for the screening of several environmental toxicants. The biosensor was constructed by immobilizing ALP in a hybrid sol-gel/chitosan film that was deposited on the surface of a screen-printed carbon paste electrode (SPE). The inhibition was measured via the catalytic hydrolysis of ascorbic acid 2-phosphate (AA2P) by the enzyme to produce ascorbic acid. Oxidation of this product was monitored amperometrically and the current change was then related to ALP activity. Toxicity of herbicides (2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), insecticides (carbofuran and α-endosulfan) and heavy metals (Hg2+, Cd2+, Ag2+, Zn2+ and Cu2+) towards the biosensor were evaluated. Various degrees of inhibition of ALP occurred when the biosensor was exposed to herbicides and heavy metals. This resulted in a lower acid ascorbic production by the enzyme from the substrate, thus a decrease in the current response of the biosensor. The herbicides 2,4-D and 2,4,5-T showed the largest inhibition effect on ALP with linear response range of 1-60 μg L-1 (R2 = 0.92). The maximum inhibitions caused by 2,4-D and 2,4,5-T were 46 and 30%, respectively. Heavy metals caused inhibition on ALP at the higher concentration range of mg L-1. Thus, the biosensor may be useful for the screening of chlorophenoxyacetic acid herbicides even in the presence of other environmental toxicants.
INTRODUCTION
The inhibition of enzymic reaction has been used very frequently to determine environmental pollutants like pesticides and heavy metals. Various enzymes such as acetyl cholinesterase (Chouteau et al., 2005; Carlo et al., 2004), alkaline phosphatase (Chouteau et al., 2005), urease (Rodriguez et al., 2004) and glucose oxidase (Malitesta and Guascito, 2005) have been reported for the construction of biosensors that used in the determination of pesticides or heavy metals.
Alkaline phosphatase, a non-specific phosphor monoesterase, is a dimeric metalloenzyme containing Zn2+ and Mg2+ ions coordinated to its active site. This enzyme has been use for indirect monitoring of pesticides or heavy metals that inhibit biocatalytic properties. Many studies have been published regarding the use of alkaline phosphatase in biosensor for the determination of pesticides or heavy metals, they included the amperometric (Mazzei et al., 2004), fluorimetric (Sanchez et al., 2004), colorimetric (Goh et al., 2005) and optical biosensors (Durrieu and Tran-Minh, 2002).
The determination of pesticides and heavy metals is important due to their toxicity and persistency in the environment. These toxic substances have been shown to be responsible for many ecological problems and damages to human health (Argese et al., 2005). Several methods can be used for pesticides and heavy metals determination in water and soil samples, which mainly involved complicated instrumentation such as chromatography or atomic absorption spectrophotometers (Amarante et al., 2003; Vink and Poll, 1996). Despite their high sensitivity and accuracy, these instrumentations are expensive, time-consuming to operate and require skillful operator. For these reasons, biosensor method is more convenient because of low cost and portability especially for rapid screening of these pollutants.
Bioelectrochemical sensor, particularly amperometric biosensor is an analytical tool that utilizes immobilized biomolecules for the conversion of a specific target analyte into electrochemically detectable products. The electrochemically active product releases from the biochemical reaction between the recognition biomolecule and the analyte will be detected by an amperometric transducer. Recent development of biosensor techniques has seen the use of immobilized enzymes, DNA (Babkina and Ulakhovich, 2004), antibody (Blake et al., 2001) and microbe (Chouteau et al., 2005). The immobilization methods for these biomolecules are usually entrapment (Andreescu et al., 2002), adsorption (Bonnet et al., 2003) and covalent binding (Liu et al., 2005).
In this study, we have reported a biosensor based on the inhibition properties of the alkaline phosphatase enzyme for the detection of pesticides as well as heavy metals. Most of the biosensors reported for such purpose so far are complicated to fabricate, which also involved tedious operating procedures. Therefore, the objective of this study was to adopt a simple mean of fabricating such a biosensor by immobilizing the alkaline phosphatase (ALP) enzyme in a sol gel/chitosan membrane and then investigate the inhibition of the enzyme by several herbicides, insecticides and heavy metals via electrochemical transduction. The membrane could be directly prepared via a one-step procedure without any complicated chemical immobilization and multilayer assembly of ALP (Chen et al., 1995). An electrochemical biosensor was constructed based on the immobilization of ALP in the sol gel/chitosan membrane. Both sol-gel and chitosan can be used as immobilization matrixes for biosensor because of their inertness and biocompatible properties (Miao and Tan, 2001).
MATERIALS AND METHODS
Reagents
Enzyme alkaline phosphatase (EC 3.1.3.1) from bovine (Sigma), tris-HCl
buffer 0.1 M, pH 8.5; ascorbic acid 2-phosphate (AA2P) (Sigma) was prepared
in distill water to give a final concentration of 0.1 M; 2, 4-dichlorophenoxyacetic
acid (2, 4-D), 2, 4, 5-Trichlorophenoxyacetic acid (2, 4, 5-T), carbofuran
and α-endosulfan were from Sigma. All metal ion solution HgNO3
(Merck), AgNO3 (Ajax Chemical), CdCl2 (General Purpose
Reagent), ZnNO3 and CuSO4 (BDH Laboratory Supplies)
were prepared in distilled and deionized water. Tetraehoxythosilane (TEOS)
was from Fluka; hydrochloric acid and glacial acetic acid from Merck.
Chitosan and MgCl2 were from Sigma.
Procedures
This research study was conducted at the laboratory of Chemical Sensor
and Biosensor Research Group, Universiti Kebangsaan Malaysia at the end
of the year 2006. The research was performed over a period of 3-4 months.
Preparation of Electrochemical Biosensor
The hybrid sol-gel/chitosan membrane was prepared by adding 2 mL of
TEOS, 3 mL of deionized water and 0.10 mL of 1 M HCl in a glass vial and
stirred for 1 h. The 1% chitosan solution was prepared in 1% acetic acid
and stirred to dissolve overnight. The hybrid of sol-gel/chitosan was
prepared by mixing both sol-gel and chitosan solution with the ratio of
9:1 (w/w). 5 microliter of hybrid sol-gel/chitosan solution which containing
ALP (2 unit m L-1) was deposited on the surface of SPE and
allowed to dry. A thin layer of film containing entrapped ALP was formed.
These electrodes were then used in the investigation of the behavior of
the immobilized ALP via electrochemical transduction.
Electrochemical Measurements
The amperometric measurements were performed using Autolab PGSTAT
12 Potentialstat/Galvanostat. The working electrode was a screen-printed
carbon electrode (SPE), which is designed by the Chem Biosensor Research
Group of the National University of Malaysia and manufactured by Scrint
Print Co, Malaysia. The amperometric measurements were performed at 0.6
V versus Ag/AgCl as the reference electrode and a platinum electrode was
used as an auxiliary electrode. A magnetic stirrer was used to stir the
solution during amperometric measurements. All experiments were performed
in 0.1 M Tris-HCl buffer, pH 8.5 containing 1 mM MgCl2.
Inhibition Studies of ALP Enzyme by Toxicants
The inhibition of the ALP enzyme was carried out by immersing the
SPE coated with enzyme film into the pesticide or metal ion standard solution
of 1-200 μg L-1 for 15 min incubation. The current Io
of the biosensor was measured before the incubation. After the incubation,
the current IA of the biosensor was measured again.
Therefore, the percentage of inhibition (% I) can be calculated based on current measured before and after exposure to the toxicants as follows:
RESULTS AND DISCUSSION
Inhibition of ALP by Pesticides
The enzyme ALP can catalyse the hydrolysis of ascorbic acid 2-phosphatase
(AA2P) to ascorbic acid. Oxidation of ascorbic acid at the electrode lead
to current changes and this is an indirect measurement of ALP activity
(Fig. 1). Whenever a sample that contains herbicide
or metal ion is added to the coupled enzyme reaction and inhibited the
biocatalytic properties of biosensor, the ascorbic acid production will
be reduced. Thus, ascorbic acid oxidation will occur at a lower rate.
By comparing the oxidation rate for acid ascorbic in an uninhibited condition,
the presence of herbicide and metal ion in a sample can be determined.
The inhibition behaviour of ALP enzymic activity can be considered as an efficient signal regarding the presence of pollutants in a sample. For 2,4-D and 2,4,5-T, both showed similar inhibition profiles (Fig. 2) and the maximum inhibition occurred at 100 μg L-1 even though the concentration studied was 1-200 μg L-1. The maximum inhibition was 46 and 30%, respectively for 2,4-D and 2,4,5-T. However, the inhibition could not achieved 100% because these herbicides are rather weak inhibitor for ALP and longer incubation time than employed here (15 min) might be needed to achieve higher level of inhibition. The linear response range of inhibition of ALP by both herbicides is the same, i.e., 1-60 μg L-1 (R2 = 0.928 and 0.913 for 2, 4-D and 2, 4, 5-T, respectively). The limit of detection (LOD) of the biosensor for 2, 4-D and 2, 4, 5-T are 0.5 and 0.8 μg L-1, respectively with the response time of less than 5 min. The reproducibility of the biosensor is within 1.0-6.0% Relative Standard Deviation (RSD). The World Health Organization (WHO) recommended that the maximum safety level of 2, 4-D and 2, 4, 5-T should not exceed 30 μg L-1 in drinking water. Therefore, the biosensor developed here can be used to screen the presence of herbicides because of its low detection limit and suitable linear response range. On the other hand, for other pesticides, a relatively lower inhibition was observed for the insecticide carbofuran and no response for α-endosulfan (Fig. 2).
| Fig 1: | The hydrolysis of ascorbic acid 2-phosphate (AA2P) by enzyme ALP to form ascorbic acid. Oxidation of ascorbic acid at the carbon paste SPE to produce dehydro ascorbic acid with the release of electrons that generate a measurable amount of current |
| Fig. 2: | The inhibition profiles of the alkaline phosphatase in the presence of each pesticide. (Experimental conditions: Substrate AA2P 80 μM, Tris-HCl buffer 0.1 M, pH 8.5, applied potential 0.6 V versus Ag/AgCl) |
Besides for the detection of herbicides, alkaline phosphatase has been used for biosensor in the detection of organophosphorus pesticides (e.g., paraoxon), where, it was first conjugated with streptavidin before immobilized in a polymer matrix on glass surface (Ayyagari et al., 1995). The synthesis of the conjugated copolymer, poly (3-undecylthiophene-co-3-thiophenecarboxyldehyde-biotin-LC-hydrozone) was complicated and the level of pesticides detected was similar to the sol-gel/chitosan based membrane biosensor reported here.
The detection limits for 2, 4-D and 2, 4, 5-T using an electrochemical immunosensor were reported to be 40 μg L-1 of 2, 4-D and 50 μg L-1of 2, 4, 5-T in both water and serum (Yulaev et al., 1996). The selectivity of this immunosensor to the herbicides was good because of the very specific binding behaviour from the conformation of the active site in the antibody molecule. However, its sensitivity was lower when compared to this study considering much effort has been made on the fabrication of this immunosensor.
Inhibition of ALP by Heavy Metals
Beside pesticides, the activity of ALP can be inhibited by some heavy
metals (Sanchez et al., 2003). The profiles of inhibition of ALP
by various heavy metals (Hg2+, Cd2+, Ag2+,
Zn2+ and Cu2+) are depicted in Fig.
3. The inhibition effect of Hg2+ on ALP was the highest
among the metal ions studied, i.e., 40 mg L-1 of Hg2+
caused an approximately 60% inhibition. The inhibition for other
heavy metals was 42% for Cu2+; 37% for Cd2+; 30%
for Zn2+ and 26% for Ag+ at 40 mg L-1
of metal concentration. Thus, the trend of response is Hg2+>Cu2+
Cd2+ Zn2+ Ag+. The metal ions Hg2+
and Cu2+ exhibited stronger inhibition on ALP because the type
of inhibition involved is non-competitive (Chen et al., 2000) and
this leads to direct inhibition of ALP without undergoing competition
with the enzyme substrate.
Chouteau et al. (2005) have reported a conductometric electrode with immobilized microalgae Chlorella vulgaris as a bioreceptor for heavy metals (Cd2+ and Zn2+) detection. The inhibition of ALP in the membrane of C. vulgaris has demonstrated a lower LOD when compared to the amperometric biosensor reported here. This is due to the higher amount of ALP from a higher concentration of algae used (4-5x10 7 cells m L-1) when compared with 2 units m L-1 of ALP employed on screen printed electrode reported here. As the enzyme concentration increases, the toxicants can be detected at lower detection limit. Besides, the algae biosensor was pre-incubated for 30-60 min in test solution, which is longer than in this study.
| Fig. 3: | The inhibition profiles of ALP by several metal ions. Experimental conditions are similar to Fig. 2 |
| Table 1: | The concentrations of various toxicants required to cause 30% inhibition to the ALP as measured by the biosensor |
At 30% inhibition level that normally considers suitable for inhibition studies, 2,4-D and 2,4,5-T herbicides showed the highest sensitivity (Table 1). The biosensor is less sensitive to heavy metals and to attain 30% inhibition, the concentrations of heavy metals need to be 1000-5000 times higher than that of herbicides. This demonstrates that the biosensor developed here has good selectivity towards the chlorophenoxyacetic acid herbicides when compared with other pesticides and toxic metals.
CONCLUSION
This study has proved that a toxicity biosensor could be constructed from the immobilization of alkaline phosphatase enzyme in a hybrid sol-gel/chitosan membrane and an electrochemical biosensor could be used to measure the toxicity caused by several toxicants. The biosensor showed inhibition by herbicides, carbofuran insecticide and toxic metals with various degrees of inhibitory response. It is most inhibited by the chlorophenoxyacetic acid herbicides and thus the biosensor demonstrated good sensitivity and selectivity for these compounds. The simple design and direct fabrication procedure of this biosensor are useful for a disposable device that can detect broad spectrum toxicity of water.
ACKNOWLEDGMENTS
The authors wish to thank the Ministry of Science, Technology and Innovation (MOSTI), Malaysia for financial support via research grants 09-03-03-0184-EA001 and 09-03-03-0006-BIOTEK. Acknowledgment to the Universiti Kebangsaan Malaysia for financial support via the Research University Operation Grant (OUP2008) and a Postgraduate Fellowship awarded to Loh Kee Shyuan.