A Cell Based Potentiometric Biosensor Biology Essay

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The characteristics of a potentiometric biosensor for the determination of permethrin in treated wood based on immobilized cells of the fungus Lentinus Sajor-Caju on a potentiometric transducer is reported. The potentiometric biosensor was prepared by immobilization of the fungus in alginate gel deposited on a pH-sensitive transducer employing a photocurable acrylic matrix. The biosensor gave a good response in detecting permethrin over the range of 1.0 - 100.0 µM. The slope of the calibration curve was 56.10mV/decade with detection limit of 1.00µM. The relative standard deviation for the sensor reproducibility was 4.86 %. The response time of the sensor was 5 min at optimum pH 8.0 with 1.00 mg/electrode of fungus Lentinus Sajor-Caju. The permethrin biosensor performance was compared with the conventional method for permethrin analysis using high performance liquid chromatography (HPLC) and the analytical results agreed well with the HPLC method (at 95 % confidence limit). There is no interference from commonly used organophosphorus pesticides such as diazinon, parathion, paraoxon and methyl parathion.


wood preservative analysis, permethrin, alginate, Lentinus Sajor-Caju

1. Introduction

Chemicals are used as protector due to their impregnation ability into the wood cell and furthermore improve their physical properties [1]. A range of different chemical treatments are able to extend the durability as well as improve the resistance to decay, insects, weather or fire [2]. Chromates 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 [3]. 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 [3]. 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 [4].

In order to achieve quality assurance of the treatment process, the quantity of the LOSP used must be sufficient as stated in the standard requirements [5]. As the main ingredient of LOSP formulations, permethrin therefore used as an indicator to determine the usage of LOSP in treated wood [6]. Up to now, the techniques employed for permethrin detection mainly involved chromatography such as High Performance Liquid Chromatography (HPLC) [7], Gas Chromatography (GC) [8-11]. Other techniques are UV-Visible Spectroscopy [12], Fourier Transform Infrared Spectroscopy [13], Flow Injection Analysis [14] and Surface Plasma Resonance [15]. Although these techniques offer reliable results, on the other hand they are non-portable devices. Therefore, simple and portable devices are being explored, as have recently been reviewed. For example, an electronic nose device developed to detect the amount of permethrin in water [16]. More recently; Kaushik (2009) has developed a nucleic acid sensor using a single strand calf thymus deoxyribose nucleic acid (ssCT-DNA) [17]. The ssCT-DNA was first immobilised onto chitosan-iron oxide nano-biocomposite film deposited on indium-tin-oxide (ITO) coated glass electrode for pemethrin detection. Shan's group has developed a sensitive immunoassay method by using enzyme-linked immunosorbent assay (ELISA) to determine permethrin in river water [18] including combining ELISA with different antibodies [19].

Therefore, alternative methods need to be explored where it should be simple, easy sensor design and of wide range applications [20-24]. Baronian et.al has suggested simpler approach by using cell-based biosensors, which demonstrated similar practice and principles of nucleic acid biosensors [25]. For permethrin, toxicity towards fungus have been studied and fungal bioassay using novel bioluminescence has been established for toxicity testing [26, 27]. The use of the fungus Lentinus Sajor-Caju has been reported for biosorption of cadmium (II) where it was entrapped in alginate gel via a liquid curing method in the presence of Cd (II) ions [28].

Until now, the fungus, Lentinus Sajor-Caju has not been employed as a cell-based biosensor for specific determination of permethrin although this fungus is known to be capable of degrading permethrin. Therefore the objective of this study is to design a new cell-based biosensor from the fungus Lentinus Sajor-Caju, which was immobilised in an alginate matrix to construct a potentiometric device consists of three layers of membranes. The biosensor can be used as a simple device to detect pyrethroid compounds, especially permethrin in treated wood.

2. Experimental

2.1 Materials

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 dioxanwere from Merck.

2.2 Fungus culture

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 sub culturing on malt dextrose agar slants. Growth medium consisted of (in g l-1 ofdistilled 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 1.0ml 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.

2.3 Electrode preparation method

0.016 g of DMPP was mixed with 0.932 mL HEMA. 1µL of this mixture was drop-coated onto Screen Printed Electrode (SPE) and then photocured for 360 s. After that, the polymer in SPE was hydrated with 0.1 mM Tris-HCL pH 7 for 15 min. Then 3.5 µL of a mixture (950 µL nBA, 1 µL HDDA, 0.8 g NaTFPB, 1 mg DMPP and 1.9 mg hydrogen ionofor) was drop-coated onto the first layer of previous polymer and photocured for 360 s. 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.

2.4 Fungus Immobilization on electrode

Alginate was used to immobilize the fungus Lentinus Sajor-Caju and it was rinsed several times with Tris-HCL buffer before mixed with alginate solution, which contained CaCl2. Finally the alginate solution was spread onto the pH sensitive membrane of the potentiometric transducer. This was then dried at 4oC overnight before measurements with permethrin were carried out.

2.5 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 buffers (PH 7) for at least 30 min. Measurements were conducted at room temperature (25OC). The EMF readings in mV were recorded after 10 min 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.

2.6 Validation of sensor response

Wood sample used in this study was Rubberwood (Hevea brasiliensis). It was treated with permethrin from 0 to 100 µM by using Vacuum Impregnation Vessel. Untreated wood sample was use as a control for the measurements. The performance of the biosensor was compared with established method using permethrin treated wood samples. In this study reference HPLC method standard method of Australian/New Zealand Standard [29] was employed. 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 samples. The HPLC condition was as follows: Detection used a photodiode detector at 260 nm, the solvent flow rate 1.5 ml/min, a loop injector of 20 μL, a column of 5 μm Luna silica (brand Phenomenex) size 150 x 4.6 mm is use to separate the cis and trans isomers.

3. Results and Discussions

3.1 Detecting principle

The biosensor consists of four layers of membranes deposited on top of an Ag/AgCl screen printed electrode (SPE). The uppermost layer is the alginate gel membrane immobilized with the fungus Lentinus Sajor-Caju (Fig 1). The use of alginate gel to entrap fungus Lentinus Sajor-Caju also been reported by Bayramoglu (2002) where the fungus was immobilized in Ca-alginate beads for the removal of Cd(II) ions from aqueous solution [28]. In a hydrophilic environment, alginate provided a stable two phase piqued system and this cause the enhancement of the operational stability of the fungi entrapped within the 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 [30]. The function of the second layer that contain hydrogen ionophore is to create a pH transducer that detects the pH changes occurred in fungus immobilized layer(Figure 1). A sample that contains permethrin first diffuses through the alginate gel membrane and reaction between permethrin and enzymes in the living fungal cells leads to formation of a weak acid. As a result, the changes in pH due to the hydrolysis of permethrin were detected by the plasticizer-free H+ selective membrane.

There is little information in permethrin pesticide biotransformation by fungi [31-32]. Lentinus Sajor-Caju is a white-rot fungus that has several extracellular enzymes for bioremediation of various xenobiotics. There is no report on the chemical reaction between permethrin and Lentinus Sajor-Caju. From the results of the permethrin biosensor response we obtained, we postulate that the reaction of permethrin with Lentinus Sajor-Caju may be similar to that reported by Liang (2005) where the production of carboxyl esterase enzyme from the fungus Aspergillus Niger ZD11 led to hydrolysis of the carboxyester linkage in permethrin to form 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid [32].

3.2 Effect of pH

The pH of the buffer could affect the potentiometric biosensor response. It was found that the pH for maximum response of the biosensor was at pH 8.0 (Figure 2). This observation is different from that reported by Bayramoglu for biosoprtion of Cd (II), Cr (VI) and uranium by Lentinus Sajor-Caju encapsulated in alginate beads [30-32]. The optimum pH in their study was at pH 5 because these metal ions are more stable under acidic conditions. In this work, slightly basic condition is required for measuring the pH change occurs from the reaction between permethrin and Lentinus Sajor-Caju since a weak acid is produced from the reaction.

3.3 Effect of buffer concentration

The best buffer concentration that yielded the highest potentiometric sensitivity slope was found at 1.0 x 10-3 M (Table 1). This is because at lower concentration of buffer, the biosensor detected more protons and this generated optimum signal. With the increase of buffer capacity, more protons will be neutralized by the buffer and the transducer will detect a smaller pH variation. Similar results were obtained by other potentiometric biosensors (Aurelia-Magdalena, 2007) [33].

3.4 Effect of fungus loading on biosensor response

The amount of fungus that can be loaded onto the electrode surface is limited by the size of the electrode area. As shown in Figure 3, the maximum weight of fungi loading was 1.00 mg/electrode. The increase of mycelia weight is proportional to the increase in the biosensor response. The response is constant after 1.00 mg/electrode because at this point all the fungus mycelia 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 nerve agents [35].

3.5 Effect of temperature on biosensor response

From Figure 4, it is clear that the temperature affected the performance of the biosensor. Maximum biosensor response was observed at lower temperatures. Above 25°C, the biosensor response is significantly reduced and this is followed by a sharp decline after 45°C. In this study, although 4°C yielded among the highest response, statistical tests using a t-test showed that there was no significant difference compared with measurements at room temperature. This observation is similar to that obtained by Karube et al (1977) for a biosensor used in the detection of BOD with fungus Pseudomonas fluorescens [36].

3.6 The dynamic response range of the biosensor

At the optimized condition of pH, buffer concentration and fungus loading, the biosensor demonstrated a near-Nernstian response to permethrin concentrations from 1 to 100µM with a sensitivity slope of 56.10 mV/decade (Figure 5), which is near to the theoretical value of 59.6 mV/decade [37]. The limit of detection, as determined from the intersection of the two extrapolated segments of the calibration curve was 1.0 µM. For the reproducibility evaluation, 10 different biosensors were exposed to 1.0 to 100.0 µM of permethrin and the RSD determined was 4.86 %. The low RSD values showed that the method was analytical acceptable [38]. The permethrin biosensor has a response time of 6 min.

3.7 Interference study

There is only slight response to organophosphorus group of insecticides that are likely to be possible interference substances. But the biosensor also responded to other pyrethroid an insecticide similar to permethrin because of the carboxyester linkage present was also subjected to hydrolysis by carboxylesterase from Lentinus Sajor-Caju (Table 2). Thus, the biosensor is selective to pyrethroid group of pesticides and not others such as OP.

3.8 Lifetime of the Biosensor

The fungal biosensor for the detection of permethrin was conducted to examine the stability and durability of the biosensor response to permethrin over a period of10-20 days. As demonstrated by Figure 6 the permethrin biosensor response remained constant over a period of more than 35 days. The reduction of the response beyond that was attributed to the deterioration of the immobilised fungus cells and thus indirectly impact on the production of H+ ions (Korpan et al. 1993) [39].

3.9 Recovery study and validation of sensor with standard method

The results from the determination of known concentrations of permethrin spiked into treated wood samples are shown in Table 3. The recovery of spike permethrin is from 90- 110 %. Thus, the biosensor is considered an acceptable method of permethrin determination in treated wood [40]. The analytical performance of the biosensor is further ascertained by comparing with analysis of permethrin using the Australian/New Zealand Standard [5].As shown in Table 4, the concentrations of permethrin determined using the sensor did not differ statistically (= 95 %) from that determined using the standard HPLC method. This demonstrates that the permethrin biosensor can be used for permethrin determination [40]. Compare with other electrochemical sensors or biosensors for permethrin, which mainly based on amperometry, the potentiometric biosensor for permethrin reported here, demonstrated a much larger linear response range of up to100 µM [17, 18, 41].

4. Conclusion

The potentiometric biosensor for permethrin based on the concept of reaction between permethrin and the fungus Lentinus Sajor-Caju cells has been successfully developed. It appeared that the action of the fungus on the permethrin produced acid that led to the response of the potentiometric pH transducer. This is probably the first potentiometric biosensor based on such a fungus developed for the determination of permethrin in treated wood. The biosensor demonstrated a large response range to permethrin when compared with many non-potentiometric devices, e.g. amperometry based sensors. The biosensor not only useful for the determination of permethrin but also other pyrethroid insecticides. There is almost no interference from other insecticides such as the organophpsporous group since the biosensor is only selective to the pyrethroid group.


The authors would like to acknowledge the Ministry of Science Technology and Innovation of Malaysia for funding this research through research grant E SCIENCE MOA (05-03-10-SF1039) and UKM for Research University funding via grant UKM-DIP-2012-11.