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Characteristics of a potentiometric biosensor for determination of permethrin in treated wood based on a three layer sandwich transducer are reported. The transducer contains a plasticizer-free methacrylic-acrylic membrane immobilized with hydrogen ionophore and a lipophilic salt. Alginate matrix was used to immobilize fungus Lentinus Sajor-Caju for the detection of permethrin. Biosensors were prepared by immobilization of the fungus on a pH-sensitive transduser. It was demonstrated that the biosensor gave a good response in detecting permethrin over the range of 1.0 µM to 100.0 µM. The slope of the calibration curve was 56.10 mV/decade with detection limit of 1.00 µM. The RSD for the sensor reproducibility was 4.86 %. The response time of the sensor was 5 min at optimum response pH 8.0 with 1.00 mg/µL of fungus Lentinus Sajor-Caju. The permethrin biosensor performance was compared with the conventional method for permethrin analysis using high performance liquid chromatography (HPLC). It was found that the concentrations of permethrin determined using the sensor was not significantly different compared to HPLC method at 95 % confidence limit. There is no interference from organophosphorus pesticide such as diazinon, parathion, paraoxon and methyl parathion.
Keywords: wood preservative, permethrin, alginate, LentinusSajor-Caju
Chemicals are used as protector due to their impregnation ability into the wood cell and furthermore improve their physical properties . A range of different chemical treatments are able to extend the durability as well as improve the resistance to decay, insects, weather or fire . Chromated Copper Arsenate (CCA) is one of the approaches which is widely been used in wood treatment. CCA becomes the best option for wood coating because of their strong chemically bond to the wood hence produces no leaching problems. There are limitations to these approach especially in solvent (water) and aesthetic value, for example. The water in CCA will wet the wood consequently subject the wood to dimensional movement upon drying . CCA also imparts a green colour to the treated wood hence, lowering the aesthetic value. In many countries, CCA is treated as unsafe and unfriendly chemical for environment . Therefore to overcome this problem, Light Organic Solvent Preservative (LOSP) is the most popular alternative treatment which is used in industrial process in preparation of softwood and hardwood products .
In order to achieve quality assurance of treatment process, the quantity of the LOSP used must be sufficient as stated in the standard requirements . As the main ingredient of LOSP formulations, permethrin therefore can be used as indicator to determine the usage of LOSP in treated wood . Up to now, the techniques employed for permethrin detection mainly involved chromatography such as High performance Liquid Chromatographhy (HPLC) , Gas Chromatography (GC) [8-11]. Other technique are UV-Visible Spectroscopy , Fourier Transform Infrared Spectroscopy , Flow Injection Analysis  and Surface Plasma Resornance . Although these techniques offer reliable results, on the other hand obviously, these techniques are time and solvent consuming, expensive and not portable devices. Therefore, low cost, simple, flexible and portable are being explored, as have recently reviewed. For example, an electronic nose device was developed to detect the amount of permethrin in water . More recent, Kaushik (2009) has developed a nucleic acid sensor using a single standard calf thymus deoxyribose nucleic acid (ssCT-DNA) . The ssCT-DNA was first immobilised onto chitosan-iron oxide nanobiocomposite film. Then, these biocomposites was deposited onto indium-tin-oxide (ITO) coated glass before could be used as pemethrin biosensor. Shan's group has developed a sensitive immunoassay by using enzyme-linked immunosorbent assay (ELISA) to determine permethrin in river water . Extending from this research, the same group has combined the ELISA with different antibody . They reported that this biomaterial was able to determine antibody that was affected by permethrin. As such the methods listed above were especially attractive but the techniques may not be applicable to all types of systems. Furthermore, the sample preparation was complicated, tedious and time consuming.
Therefore, alternatives methods need to be explored where it should be simple, easy functionalization and wide range applications [20-24]. Baronian et.al have suggested simpler approached by using cell-based biosensors which demonstrated similar practice and principles of nucleic acid biosensors . In early 80s, a study on permethrin toxicity effects in ten different fungi was made . Prior to this study, a novel bioluminescence-based using fungal bioassay has been established for toxicity testing . The majority of literature describing biosensors and chemical sensors has focus on nucleic acid-based or nose device sensor. On the other hand, no literature was found describing cell-based biosensor using a specific fungus, Lentinus Sajor-Caju. Also none of the work reported in the literature has looked at the possibility of using fungi cell-based biosensor for permethrin detection. Gulay Bayramoglu (2002) use fungus Lentinus Sajor-Caju as biosorption of cadmium(II) . The Lentinus Sajor-Caju was entrapped into alginate gel via a liquid curing method in the presence of Cd(II) ions.
Therefore the objective of this study is to explore the development of cell-based biosensor using fungus Lentinus Sajor-Caju. In this work, Lentinus Sajor-Caju is immobilised in an alginate matrix within three layers of the systems. A stable systems and results are expected and moreover, this biosensor can be used as a simple device to detect permethrin in treated wood.
Materials and methods
Chemicals used in this study were: 2-hydroxyl ethyl methacrylate (HEMA), 2,2-dimetoxy-phenyl-acetophenone (DMPP), n-butyl acrylate (nBA), sodium tetrakis [3,5-bis (trifluoromethyl) phenyl] borate (NaTFPB), poly (2-hydroxyl ethyl methacrylate) pHEMA), 1,6-hexanadiolacrylate (HDDA), tris (hydroxymethyl) aminomethane (Tris-HCl), sodium alginate, hydrogen ionophore, citrate buffer, permethrin and dioxsan were from Merck.
Forays were conducted by Wood Mycology Unit of Forest Research Institute Malaysia (FRIM) in 2006 and 2007 with the aim to study the occurrence of Lentinus species and population in the Malaysian forest concentrating only in Peninsular Malaysia. A number of Lentinus species were found at various locations in the forests with Lentinus Sajor-Caju showing the widest distribution in the forays. 15 specimens were collected in Endau-Rompin National Forest, which were identified only up to genus level. Lentinus Sajor-Caju was found as the most frequent species collected in this study.
Lentinus Sajor-Caju was maintained by subculturing on malt dextrose agar slants. Growth medium consisted of (in g l-1 of distilled water); D-glucose, 10.0; KH2PO4, 20.0; MgSO4.7H2O, 0.5; NH4Cl, 0.1; CaCl2.H2O, 0.1; thiamine, 0.001; and it was supplemented with 1.0 ml of trace element solution (containing g l-1;nitrilotriacetate, 1.5; NaCl, 1.0; MnSO4.H2O, 0.5; FeSO4.7H2O, 0.1; ZnSO4, 0.1; CaSO4, 0.01;CuSO4.5H2O, 0.01; H3BO3, 0.01; NaMoO4.2H2O, 0.01).The pH of the medium was adjusted to 4.5, which was the optimum growth pH of Lentinus Sajor-Caju.
Electrode Preparation Method
0.932 mL HEMA was added to 0.016 g of DMPP. 1µL of this mixture was drop coated onto Screen Printed Electrode (SPE) and then fotocure for 360 second. After that, the polymer in SPE was hydrated with 0.1 mM Tris HCL pH 7 for 15 minute. Then dropcoat 3.5 µL of a mixture (950 µL nBA, 1µL HDDA, 0.8 g NaTFPB, 1mg DMPP and 1.9 mg hydrogen ionofor) onto the first layer of previous polymer. Fotocured for 360 second. The response of the sensor to hydrogen ions was tested with a double junction Ag/ AgC1 electrode as the reference electrode. The electrode and sensor were connected to an Orion ion meter where the difference in the potential of the cell (electromotive force, EMF (mV)) was recorded when a stable value was reached. The sensor was examined with 0.l mM Tris HCl buffer at pH 4.0 -9.0. The pH of each buffer solution was measured with a pH electrode before use. The EMF response of the test cell was plotted against the logarithmic concentrations of the test solutions according to the Nernst equation.
Alginate was used to immobilize fungus Lentinus Sajor-Caju. Lentinus Sajor-Caju was wash few times with Tris HCL buffer and then mixed with Alginate solution which contains CaCl2 before it was spread onto membrane of the pH transducer. This was then dried at 4oC for overnight before can be use for measurements with permethrin were carried out.
Evaluation of biosensor response
Permethrin solutions in the range of 0.1 to 0.1 mM were prepared in Tris-HCl buffer (0.l mM pH 7). Measurements were carried our as described above. Before use, the electrode was equilibrated in 0.1 mM Tris HCI buffer (PH 7) for at least 30 minutes. Measurements were conducted at room temperature (25OC). The EMF readings in mV were recorded after 10 minutes and were plotted against the logarithmic of permethrin concentration to establish the calibration curve. The optimum pH, effect of buffer concentrations, enzyme optimization, effect of temperature, dynamic response range, response time of the sensors, repeatability, reproducibility, lifetime and interference characteristics and the sensor regeneration capability were evaluated.
Possible interference of the sensor from cypermethrin, deltamethrin, diazinon, parathion, paraoxon and methyl parathion was investigated.
Wood sample were used in this study was Rubberwood (Hevea brasiliensis). This wood samples treated with concentration range of permethrin from 0 to 100 µM by using Vacuum Impregnation Vessel. Untrated wood use as a control for the measurements.
Validation with establish method
The performance of developed biosensor was compare to establish method. In this study we use British Standard, DD 257-3:2003 Method 1: HPLC as reference. The organic solvent (n-hexane) and mobile phase (99.5% n-hexane: 0.5% THF) were used for separation and determination of cis and trans isomers of permethrin in treated wood. There chemicals are determined in HPLC with Photodiode Detector at 260 nm, at the solvent flow rate 1.5 ml/min, a 20 μl loop injector, a column of 5 μm Luna silica (brand Phenomenex) size 150 x 4.6 mm is use to separate the cis and trans isomers.
Results and Discussions
The developed biosensor consists of four layer of membrane on top of Ag/AgCl Screen Printed Electrode (SPE). Outer is the crucial one where fungus Lentinus Sajor-Caju in alginate gel. The use of alginate gel to entrap fungus Lentinus Sajor-Caju also been reported by Bayramoglu (2002) where Lentinus Sajor-Caju was immobilized into Ca-alginate beads for the removal of Cd(II) ions from aqueous solution . In hydrophobic ambient, alginate provided a stable two phase piqued system and this cause the enhancement of the operational stability of fungi entrap within alginate layer. Beside alginate other organic and inorganic materials such as polypyrrole films, biocompatible synthetic latex and laptonite also has been reported as suitable matrix for fungi entrapment . The function of second layer that contain hydrogen ionofor is to work as pH transducer and detect the pH changes occurred in third layer. This response then optimize transfer to Ag/AgCl electrode with the existence first membrane layer that contain HEMA and DMPP polymer. With this system (Figure 1), sample that contain permethrin first diffuse through the alginate gel membrane and reaction between permethrin and enzyme form weak acid.
Figure 1 : The design of permethrin biosensor
As a result, the changes in pH due to the hydrolysis of permethrin were detected by plasticizer-free H+ selective membrane. There is little information in permethrin pesticide biotransformation by fungi [30-31]. Lentinus Sajor-Caju is a white-rot fungus and has several extracellular enzymes for bioremediation of various xenobiotics. There is no reported exactly chemical reaction between permethrin and Lentinus Sajor-Caju. Therefore we are searching the similar fungi in the white-rot fungus group that can react with permethrin. Liang (2005) reported the production of carboxyl esterase enyme from fungus Aspergillus Niger ZD11. This enzyme will hydrolyses carboxyester linkage in permethrin compound to form 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid .
Effect of pH
The pH of the buffer will affect the developed biosensor. It was faund that the optimum pH for this biosensor are at pH 8.0 (Figure 2). This finding is different from Bayramoglu finding reported in research of biosoprtion of Cd(II), Cr (VI) and uranium by Lentinus Sajor-Caju encapsulated in alginate beads [29-31]. The optimum pH in their study was found at pH 5. The different of this situation is because of the different ambient and technique of measurement. In this work, slightly bes pH more suitable to measure the change occurs from the reaction between permethrin and Lentinus Sajor-Caju. The weak acid produce from reaction easily measured in neutral condition compared to acidic condition. While in the biosorption only measured the adsorption of the metals onto the beads and do not measured the amount of H+ produce from the reaction [29-31].
Figure 2. Effect of pH on permethrin biosensor response. The permethrin concentrations range from 2.00x10-8 M to 2.51x10-3 M
Effect of buffer concentration
The optimum buffer concentration was found at 1.0 x 10-3 M. This is because at lower concentration of buffer, the biosensor senses a bigger amount of proton and this generated optimum detection of the biosensor (Table 1). With the increase of buffer capacity, more protons will be neutralized by the phosphate present in the buffer and the transducer will sense a smaller pH variation. Similar result was obtained by other potentiometric biosensor (Aurelia-Magdalena, 2007) .
Table 1. Effect of buffer concentration on permethrin biosensor response. The permethrin concentrations range from 1.0 to 80.0 µM
Buffer Concentration (M)
1 x 10-1
1 x 10-2
1 x 10-3
1 x 10-4
1 x 10-5
Effect of fungus loading
The constrain that need to be consider is the space in the screen peinted electrode. Limited space will affect the fungus loading because the size of the Lentinus Sajor-Caju mycelia. More weight means bigger size of the mycelia. As shown in figure 3, the optimum weight of fungi loading was 1.00 mg/ul of alginate. The SPE used only can occupy the mycelia up to 2.00 mg/L. The weight more than this will cause the mycelia spread outside of the ring and this will affect the reading. The increase of mycelia weight is proportional to the increase of the biosensor response. The response is constant after 1.00 mg/ul because at this point all the fungus mycela ia already reacted with permethrin and permethrin is excess. Ashok Mulchandani (1998) also reported similar finding in the construction of potentiometric microbial biosensor for the direct measurement of organophosphate (OP) nerve agents .
Figure 3. Effect of fungus loading on the biosensor response. Permethrin concentrations 2.00x10-8 M to 2.51x10-3 M
Effect of temperature
Studies carried out to determine the effect of temperature either fungal biosensor developed can be influenced by changes in temperature. From the Figure 4 it was clearly seen that the temperature affects the performance of the fungus biosensor. Optimal detection conditions were observed at low temperatures. Temperature increase after 25°C resulted biosensor sensitivity is significantly reduced and this was followed by a sharp decline after the temperature sensitivity of 45°C. In this study, although the temperature 4°C gave a response to the high sensitivity, statistical tests using a t-test found that there is no significant difference if the measurements were performed at room temperature. These results found to be similar with the obtained by by Karube et al (1977) in his study developed a biosensor for the detection of BOD by using Pseudomonas fluorescens fungi .
Figure 4 Study the effect of temperature on the biosensor response. Permethrin concentration range tested was from 2.00x10-8 M to 2.51x10-3 M.
At the optimized condition of pH, buffer concentration and fungus loading, the biosensor developed produce a near-Nernstian response to the permethrin concentrations from 1 µM to 100 µM with a slope of 56.10 mV/decade (Figure 5) which is near to the theoretical value of 59.6 mV /decade. This linear relationship between the measured emf (E, in mV) and permetrin concentration derived from equation:
E = E0 - P log [Permethrin Concentration] (1)
Where E0 is the formal cell potential and P represents the Nernst coefficient (59.16 mV/decade, at 250, for monovalent ions) . The limit of detection, as determined from the intersection of the two extrapolated segments of the calibration graph was 1.0 µM. Other researcher also get the result near Nernstian response in the potentiometric study using cell based biosensor . For the reproducibility evaluation, 10 different sensors were exposed to 1.0 to 100.0 µM of permethrin and the RSD determined was 4.86 %. The low RSD show that the method was analytical acceptance . This constructed biosensor takes about 6 minute to achieve constant value.
Figure 5. The dynamic range response of a biosensor towards permethrin concentrations from 1.0 to 100.0 µM had in optimized conditions.
Lifetime of the Biosensor
Life time study of fungal biosensor for detection of permethrin conducted to examine the stability and durability of the biosensor was constructed. Evaluation is done by measuring the biosensor response permethrin over time intervals between ten to the twenty days. From Figure 6 is was found that the measurement after storage affect permethrin biosensor response. This happens because when the biosensor is stored, cells fungus has undergone biological changes such as decreased enzymes reactivity in it and this will inhibit hydrolysis reaction by enzymes in the cell and thus have an impact on the production of H+ ions (Korpan et al. 1993) . The study showed that the developed fungus biosensor has could provide acceptable analytical performance when stored for twenty days as still give a stable reading
Figure 6. Lifetime of a biosensor towards permethrin concentrations from 1.0 to 100.0 µM had in optimized conditions.
From Table 2, it shows that other chemicals in pyrethroid group give similar result with the permethrin. These means that Lentinus Sajor-Caju is not specific towards insecticide in pyrethroid groups. This is because all insecticide in the pyrethroid group have carboxyester linkage and subjected to be hydrolyse by carboxylesterase in Lentinus Sajo Caju.
Table 2. Interference study of a biosensor towards interference chemicals concentrations from 1.0 to 30.0 mg/L
6.10 to 11.90 µM
5.20 to 11.10 µM
Permethrin concentration in treated wood is measured by using developed biosensor. The result are shown in Table 3. The recovery of spike samples is from 90 % to 110 %. This means developed biosensor gives good analytical determination of permethrin in treated wood .
Validation with Standard Method
The analytical performance of the sensor for the analysis of permethrin was compared to that of British Standard, DD 257-3:2003 Method 1: HPLC . As shown in Table 4, the concentrations of permethrin determined using the sensor did not differ statistically (ï¡ value = 95 %) from that determined using the HPLC method. This demonstrates that the permethrin sensor developed in this study can be used for permethrin determination with confidence .
Table 3. Recovery study of the biosensor
Determined concentration (mg/kg)
(n = 7)
9.0 ± 2.3
22.0 ± 3.0
* using Rubberwood sawdust
Table 4. Validation study of the biosensor (Determination of permethrin in Rubberwood)
Standard Method using HPLC (mg/kg) (n=3)
Biosensor method (mg/kg) (n=3)
A Lentinus Sajor-Caju cell-based Potentiometric biosensor for determination of permethrin in treated wood was successful produced and characterised. The main advantage of this biosensor is its construction simplicity and low cost. Optimum working conditions were pH 7 and temperature of 25 -C. A response of about 5 min was registered for Lentinus Sajor-Caju immobilised on a three layer sandwich transducer. The interference of organophpsporous insecticide in permethrin determination is not significant whreas there a significantly interference from pyrehthroid group. A linear calibration plot was recorded for permethrin concentration and giving response near the
Nernstian. Compared to other study, this biosensor produce lightly higher range of concentration permethrin up to 100 mg/L than 2 mg/L reported previously.
The authors would like to acknowledge the Ministry of Science Technology and Innovation of Malaysia for funding this research through research grant E SCIENCE 05-03-10-SF0036.