TMCnet News
A Portable Pesticide Residues Detection Instrument Based on Impedance Immunosensor [Sensors & Transducers (Canada)](Sensors & Transducers (Canada) Via Acquire Media NewsEdge) Abstract: In this paper, a design of portable pesticide residues detection instrument was presented based on an impedance immunosensor. The immunosensor exploited the novel multilayer films based on Au nanoparticles (AuNPs) and polyaniline/carboxylated multiwall carbon nanotubes-chitosan nanocomposite (PANI/MWCNTs/CS). The detection principle of the instrument was based on the electrochemical characteristic of antigen-specific antibody immune response. With a stronger signal generated from the antigen-specific antibody immune response, the signal detection circuit was designed more easily. We integrated immunosensor and signal detection circuit to fabricate pesticide residues detection instrument. This proposed instrument could realize the rapid detection of pesticide residues in fruits and vegetables with automatic data processing and presented the result on the spot. The impedance test error was less than 5 %. The results showed that the proposed instrument had a good consistence compared with the traditional analytical methods. Thus, it would be a promising rapid detection instrument for pesticide residues in agricultural products. Copyright © 2014 IFSA Publishing, S. L. Keywords: Pesticide residues, Immunosensor, Impedance detection circuit, Portable instrument, Antigen-specific antibody immune response. 1. Introduction Pesticide has been widely used in the field of modem agricultural production, which can effectively control pests to improve crop yields. In particular, organophosphorus compounds are widely applied to improve the output of agricultural production [1]. The extensive utilisation of pesticides ensures the stability of crop yields, and also brought a lot of environmental problems and cause severe impairment of human health. At the present, classical chromatographic analysis, such as gas chromatography (GC), high-pressure liquid chromatography (HPLC), capillary electrophoresis (CE) and mass spectrometry (MS), are very sensitive and standardized techniques [2, 3]. These methods are very sensitive and reliable; however, they used a large of reagent, professional operation, time-consuming and expensive. For these reasons, the development of rapid and efficient monitoring methods for recognitive and quantitative detection of the presence of pesticide residues in the food is becoming more and more important. In order to avoid potential harm for human health, detecting pesticide residues in vegetables and fruits on-site is necessary [4]. Electrochemical immunosensors based on the high specificity of hapten (pesticides such as carbofuran, chlorpyrifos) and antibody (Ab) interactions have been used to detect or quantify a specific pesticide. Compared with conventional methods for the determination of carbofuran, electrochemical immunosensors have many advantages, including simple instrumentation, easy operation, rapid response, high sensitivity, selectivity and high compatibility with advanced nanotechnology and micromachining technologies [5-7]. It has been reported that biosensor measurements are a good method for rapid detection of pesticide residues. Immunosensors, which combine the selectivity provided by immunological interactions, are also being proposed and proving to be powerful analytical devices for the monitoring of organic pollutants in food and the environment [8]. Immunosensors show the ability of high specificity, low cost, and rapid analysis. The detection principle is the antigen-specific antibody immune response, with antibody as recognition element, by means of immobilization of the antibody binding to the receptor surface [9-11]. The immune responses result in the changes of physical and chemical signals. This signal can be quantified and handled through the instrument. By processing output, the pesticide concentration is ultimately obtained. Electrochemical analysis instrument is a universal instrument, not a specific detection instrument [12]. Biosensor technologies have been developed with amperometry, piezoelectricity, and optical waveguide, Scognamiglio, et al. have designed and developed one miniaturized multifunction biosensor array system for the detection of endocrine disrupting chemicals which equipped with optical excitation and detection, current measurement and flow control systems [13]. Grossi et al. have developed an embedded portable biosensor system for bacterial concentration [14]. Schöning et al. have fabricated a flow-injection system with dual amperometric and potentiometric organophosphorus pesticides (OP) biosensors for the simultaneous and rapid measurements of OP compounds was described [15]. All these methods are highly competitive with traditional analytical techniques in terms of shorter time response and lower cost, but they lack sufficient sensitivity and repeatability, on the other hand, they are unsuitable for industrial or commercial applications due to rather complex procedures. Here, an impedance circuit was designed to construct a portable detection instrument based on the impedance immunosensor. The immunosensor exploited the novel multilayer films based on AuNPs and PANI/MWCNTs/CS. PANI-coated MWCNTs were prepared by in situ chemical polymerization and carboxylated MWCNTs played an important role in obtaining the thin and uniform PANI resulting in the improved immunosensor response. AuNPs were used as a linker to immobilize chlorpyrifos antibody. The immunosensor and the impedance detection circuit were integrated to achieve the integration detection system, which met the requirements of detecting the pesticide residues fast and on-line test. A good precision, high stability and accuracy of the pesticide residues detection instrument in standard pesticide solutions were investigated. 2. Experimental and Methods 2.1. Schematic Drawing of Detection Device Structure The process of impedance detection could be realized in this way: firstly, the impedance signal strength was changed when antigen-specific antibody immune response had happened [16]. Secondly, AD5933 generated excitation signal which applied to the test impedance, and collected response signal. Thirdly, digital processing DFT module outputted the result to microcontroller. Finally, the microcontroller would compare result with the previous impedance value and get the difference. Compared the rate of change with the standard curve, the microcontroller would output the conclusions about pesticide concentration. The consequence of the detection was displayed on the LCD screen under the control of microchip. The schematic drawing of designed detection system based on the microchip was shown in Fig. 1. 2.2. The Design of Power Supply Circuit Because the precision of voltage source played an important role in the accuracy of the entire experiment, the power circuit part adopted the threeterminal voltage regulator. The power supply circuit chose 5 V, as the chips were used under 5 V rated voltage. The overall design concepts were: 1) The power transformer reduced the AC voltage to AC voltage required. 2) After the step-down, only one-way direct current (DC) was obtained through the rectifier circuit, but its amplitude changes were obvious. 3) By passing filter circuit, pulsating larger DC was converted a smooth, small ripple of direct current. 4) The voltage regulator could make filtered DC voltage to become a stable DC voltage without outside influence. The design of power supply circuit was represented in Fig. 2. 2.3. The Design of Impedance Core Detection Circuit The design of core detection circuit was based on the AD5933 chip design. The AD5933 is a high precision impedance converter system solution which combines an on board frequency generator with a 12 Bit 1 MSPS ADC. The frequency generator allows an external complex impedance to be excited with a known frequency. The response signal from the impedance is sampled by the ADC and DFT processed by a DSP on board The DFT algorithm returns a Real (R) and imaginary (I) data word at each output frequency [17]. In the design detection circuit, AD5933 chip under the control of the microcomputer produced excitation voltage. The generated excitation signal was applied to the test impedance. After that, ADC module would sample the response signal, and transported it to the DFT digital processing module. The DFT digital module processed the signal and outputted the result to the microcontroller. Then the microcontroller calculated and calibrated results. Compared with the previous impedance value, the microcontroller would get the difference. Finally, the consequence of the detection was displayed on the LCD screen under the control of microchip. The design of core detection circuit was represented in Fig. 3. 2.4. The Design of Display Module Display module design was based on LCD 12864 screen. It was equipped with the performance of flexible interface; simple; convenient operation instruction; lower power consumption; simple hardware circuit structure and concise display program. Therefore, it could be suitable to make the design to achieve miniaturization and portable instrument. In our design display module, liquid crystal display data interface connected with MCU PO mouth through the pull-up resistor. The design of display module was represented in Fig.4. 2.5. The Whole Instrument Synthesis The pesticide residues detection instrument was integrated by immunosensor, cell and signal detection circuit. The cell was a place for pesticides and antibody response. The immunosensor was used to fix antibodies. The detection circuit was applied to collect and process data. 3. Experimental 3.1. Detection Circuit Performance Evaluation In order to test the accuracy of the detection circuit, we carried on the following experiment. First, we chose the five different sizes of standard resistance, and divided them into five groups. Then, a multimeter measured them for 5 times respectively, and an average impedance of each group was got. Third, we used the detection circuit to complete the detection according to above-mentioned, and got another impedance value of each group. 3.2. The Preparation Process of Immunosensor A novel multilayer film based on Au nanoparticles (AuNPs) and polyaniline/carboxylated multiwall carbon nanotubes-chitosan nanocomposite (PANI/ MWCNTs/CS) was exploited to fabricate a highly sensitive immunosensor for detecting chlorpyrifos. PANI-coated MWCNTs were prepared by in situ chemical polymerization and carboxylated MWCNTs played an important role in obtaining the thin and uniform coating of PANI resulting in the improved immunosensor response. AuNPs were used as a linker to immobilize chlorpyrifos antibody 6 pi PANI/MWCNTs/CS dispersion was dropped onto the surface of the GCE (denoted as PANIMWCNTs/CS/GCE). Then the prepared electrode was rinsed with distilled water to remove loosely adsorbed PANI/MWCNTs/CS. The AuNPs was coated on the electrode soon afterwards (denoted as AuNPs/PANI/MWCNTs/CS/GCE). The AuNPs were adsorbed onto the PANI/MWCNTs/CS/GCE by chemisorptions-type interactions between NH2 group and AuNPs. Subsequently, the modified electrode was immersed in the anti-chlorpyrifos anti-body solution at 4 °C for about 12 h (denoted as antichlorpyrifos / AuNPs / PANI / MWCNTs / CS/GCE). AuNPs, for antibody immobilization, could improve the electrochemical signal and adsorption capacity of antibody, and thus enhanced the detection sensitivity. At last the electrode was incubated in 5 % BSA solution for about 1 h in order to block possible remaining active sites and avoid the nonspecific adsorption (denoted as BSA/antichlorpyrifos/AuNPs/PANI/MWCNTs/CS/GCE). The finished immunosensor was stored above the 0.1 M PBS at 4 °C when not in use [18]. 3.3. Measurement Procedure The pretreated immunosensor could exhibit a good connection with rapid detection circuit, and immersed in electrochemical reaction cell containing 9mL PBS (0.1 mol/L, pH 7.5) electrolyte. Turned on the power switch, the detection circuit was made to running. After the impedance had reached a steady-state and the response impedance value was recorded as Zo. For measurement of pesticides, the pretreated immunosensor was immersed in standard pesticides solutions with 5 pg/mL concentration for 25 min, and then transferred to the electrochemical cell. Finally pressed start testing switch and the response impedance value was recorded as Z\. The value of inhibition ratio was given by following formula: Inhibition ratio (%) = (Zi - Zo)/Zox 100%, (1) where Zo is the magnitude of impedance before immunosensor was exposured to pesticide, and Zi is the magnitude of impedance after immunosensor was exposured to pesticide. 4. Results and Discussion 4.1. The Test of Instrument's Stability Compared the error between the two values, we got a relative error rate. Results were summarized in Table 1. The impedance test error was less than 5 % in this detection circuit. The results showed that the detection circuit was stable and the test error was acceptable. 4.2. The Detection of the Chlorpyrifo Standard Pesticide To further demonstrate the practicality of the proposed detection instrument, we tested 8 groups of 50 ng/mL concentration of standard pesticide solutions. Results were summarized in Table 2. It indicated that the proposed detection circuit was accurate, reliable and reproducible. It can be used for directing analysis of practical samples. The linear regression equations for inhibition rate and the concentration of pesticide were y=8.8534 lgC (pg/mL) + 10.736 (R2 = 0.9936). Where y was the value of inhibition rate and C was the concentration of pesticide. The overall performance of the present instrument showed the capability of the pesticide residues detection with good sensitivity and high practical value. From the Table 1, it can be seen that the impedance test error was below 5 %. We can draw the pesticide added standard recovery was more than 70 % from Table 2. The accuracy of measurement was acceptable and could meet the requirements of rapid detection of pesticide residues. 4.3. Comparison with the Existing Rapid Instruments Currently, the portable pesticide residue instruments on the market are using the enzyme inhibition method, using the organophosphorus pesticide can inhibit the activity of acetylcholinesterase principle. Electric current produced by the reaction of enzyme and substrate is very small. After the enzyme contacted with pesticides, the current would be smaller. It was hard to guarantee accuracy of measurement because the current was highly susceptible to interference. In order to reduce the error, we need to do a lot of complex work. Even so, the measurement precision was still difficult to guarantee. The principle of the pesticide residues detection instrument was based on impedance measurement. After pesticide reacted with antibody, the impedance value increased with the easier measured. NC-800 and PR-3, the portable pesticide residues instruments, are applied widely. However, a longer time needed for their detection and they could only provide a qualitative test. If we wanted to treat liquid measurement for quantitative analysis of the pesticide concentration, we would also need to other laboratory analysis method. The portable pesticide residues detection instrument which we designed could be on-site rapid detection and reflected the test results intuitively. The pesticide adding standard recovery was more than 70 %. It was acceptable and could meet the requirements of rapid detection of pesticide residues. 5. Conclusions In this paper, we developed a miniaturization and portable pesticide residues detection instrument based on highly sensitive impedance immunosensor for chlorpyrifos residues detection. The immunosensor was prepared by immobilizing antibody onto working electrode surface modified by Au nanoparticles (AuNPs) and polyaniline/carboxylated multiwall carbon nanotubes-chitosan nanocomposite (PANI/MWCNTs/ CS). The instrument was integrated by the impedance immunosensor and signal detection circuit, which has been tested on standard resistance and chlorpyrifos concentration. The results showed that the proposed instrument was reliable, and could meet the rapid pesticide residues detection requirements. It would be a promising rapid detection instrument for pesticide residues in agricultural products. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 30972055, 31101286), Agricultural Science and Technology Achievements Transformation Fund Projects of the Ministry of Science and Technology of China (No. 2011GB2C60020) and Shandong Provincial Natural Science Foundation, China (No. Q2008D03). References [1] . M. Angel González-Curbelo, M. V. Herrera-Herrera, L. M. Ravelo-Pérez, L. J. Hemández-Borges, Sample-preparation methods for pesticide-residue analysis in cereals and derivatives, TrAC Trends in Analytical Chemistry, Vol. 38,2012, pp. 32-51. [2] . S. Laschi, D. Ogoñczyk, I. Palchetti, M. Mascini, Evaluation of pesticide-induced acetylcholinesterase inhibition by means of disposable carbon-modified electrochemical biosensors, Enzyme Microbial Technology, Vol. 40, Issue 3,2007, pp. 485-489. [3] . A. N. Ivanov, L. V. Lukachova, G. A. Evtugyn, E. E. Karyakina, S. G. Kiseleva, H. C. Budnikov, A. V. Orlov, G. P. Karpacheva, A. A. Karyakin, Polyaniline-modified cholinesterase sensor for pesticide determination, Bioelectrochemistry, Vol. 55, Issue 1-2,2002, pp. 75-77. [4] . E. Mauriz, A. Calle, A. Montoya, L. M. Lechuga, Determination of environmental organic pollutants with a portable optical immunosensor, Talanta, Vol. 69, Issue 2,2006, pp. 359-364. [5] . E. P. Syrago-Styliani, G. Evagelos, T. Anthony, A. S. Panayotis, Gas chromato-graphic-tandem mass spectrometric method for the quantitation of carbofuran, carbaryl and their main metabolites in applicators' urine, Chromatogr., 1108, 2006, pp. 99-110. [6] . J. N. Zhu, M. J. Jin, W. J. Gui, Y. R. Guo, R. Y. Jin, C. M. Wang, Development of a direct competitive enzyme-linked immunoassay for carbofuran in vegetables, Food Chem., Vol. 107, 2008, pp. 1737-1742. [7] . T. Liu, H. C. Su, X. J. Qu, P. Ju, L. Cui, S. Y. Ai, Acetylcholinesterase biosensor based on 3-carboxyphenylboronic acid/reduced grapheme oxide-gold nanocomposites modified electrode for amperometric detection of organophosphorus and carbamate pesticide, Sens. Actuators B: Chem., Vol. 160,2011, pp. 1255-1261. [8] . X. Sun, Y. Zhu, X. Y. Wang, Amperometric Immunosensor Based on a Protein A/Deposited Gold Nanocrystals Modified Electrode for Carbofuran Detection, Sensors, Vol. 11,2011, pp. 11679-11691. [9] . M. Pohanka, P. Skladai, Electrochemical biosensor-principles and applications, Appl. Biomed, Vol. 6, 2008, pp. 57-64. [10] . S. Kumar, Aaron J., Sokolov K., Directional conjugation of antibodies to nanoparticles for synthesis of multiplexed optical contrast agents with both delivery and targeting moieties, Nat. Protoc., Vol. 3,2008, pp. 314-320. [11] . P. Sharma, K. Sablok, V. Bhalla, C. R. Suri, A novel disposable electrochemical immunosensor for phenyl urea herbicide diuron, Biosens. Bioelectron., Vol. 26, 201 l,pp. 4209-4212. [12] . X. Sun, X. Y. Wang, S. Y. Du, Y. Zhu, A Weak Current Detection Circuit design of Organophosphorous Pesticides Rapid Detection Portable Instrument, Advanced Materials Research, Vol. 403-408,2012, pp. 2569-2572. [13] . V. Scognamiglio, I. Pwzzotti, G. Pezzotti, J. Cano, I. Manfredonia, K. Buonasera, F. Arduini, D. Moscone, G. Palleschi, M. T Giardi, Anal. Chim. Acta., Vol. 751,2012, pp. 161-170. [14] . M. Grossi, M. Lanzoni, A. Pompei, R. Lazzarini, D. Matteuzzi, B. Riccö, An embedded portable biosensor system for bacterial concentration detection, Biosensors and Bioelectronics, Vol. 26, 2010, pp. 983-990. [15] . M. J. Schöning, R. Krause, K. Block, M. Musahmeh, A. Mulchandani, J. Wang, A dual amperometric/potentiometric FIA-based biosensor for the distinctive detection of organophosphorus pesticides, Sens Actuators B: Chem., Vol. 95, Issue 1-3,2003, pp. 291-296. [16] . X. X. Sun, X. Sun, X. Y. Wang, A Design of Portable Pesiticide Residue Detection System Based on the Enzyme Electrode, Sensors & Transducers, Vol. 149, Issue 2,2013, pp. 116-122. [17] . J. Li, S. L. Chen, S. J. Jin, Design and implementation based on AD5933 impedance analyzer, Modem Scientific Instruments, 2009, No. 2, pp. 28-56. [18] . X. Sun, Q. Lu, X. Y. Wang, A Novel Immunosensor Based on Au Nanoparticles and Polyaniline/ Multiwall Carbon Nano-tubes/Chitosan Nanocomposite Film Functionalized, Nano-Micro Interface, Vol. 72,2013, pp. 191 -201. Jiang Ding, Yemin Guo, Huiying Jia, * Lu Qiao, Xia Sun, Xiangyou Wang School of Agriculture and Food Engineering, Shandong University of Technology, No. 12, Zhangzhou Road, Zibo 255049, P. R. China Tel.: +86-533-2786558, fax: +86-533-2786558 Received: 7 April 2014 /Accepted: 30 May 2014 /Published: 30 June 2014 (c) 2014 IFSA Publishing, S.L. |