SYNTHESIS AND CHARACTERIZATION OF PANI-Fe0.5-(Al0.5, Ni0.5, Mn0.5) NANOCOMPOSITE THIN FILMS FOR LEPTOSPIRA BIOSENSOR
Jamal Jurait1, Huda Abdullah1, Norshafadzila Mohammad Naim1 and Siti Khairani Bejo2
1Department of Electrical, Electronic and System Engineering, Faculty of Engineering and Built Environment,
Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia
2Department of Veterinary Pathology & Microbiology, Faculty of Veterinary Medicine, Universiti Putra Malaysia,
43400 UPM, Serdang, Malaysia
Corresponding author: [email protected]
ABSTRACT
Leptospira bacteria started from the urine of rats to the water reservoirs and spread out the water environment became polluted. The main cause of Leptospirosis disease is the pollution in lake and mud. This paper discusses the sensitivity of thin films to detect Leptospira bacteria in water environments. The thin films were produced by using the sol-gel method which used the metals: Ferum (Fe), Aluminum (Al), Nickel (Ni), Manganese (Mn) which have been doped with the polyaniline (PANI). Impedance spectroscopy electrochemistry (EIS), Current voltage (I-V) characteristic, and UV-Vis spectroscopy analysis have been used to identify their performance and criteria of the PANI-Fe0.5-(Al0.5, Ni0.5, Mn0.5) Nanocomposite Thin Film.
Keywords: Leptospira bacteria; Polyaniline (PANI); Ferum (Fe); Aluminum (Al);
Nickel (Ni); Manganese (Mn); Sol-gel method
INTRODUCTION
Leptospirosis disease was discovered and became a global health issue in the tropical rainforest and subtropical country like Indonesia, Malaysia, and Brunei. Generally, Leptospirosis was a zoonotic disease that was obtained from animals as a model carrier and intermediate hosts, which considered emerging global public health [1,2]. The effect of the Leptospirosis disease was kidney failure and internal organ of human body.
The infectious disease caused by the genus pathogenic strain of Leptospira interrogans, that the genus could be delivered directly or indirectly from animal to human [3]. The main cause of Leptospirosis disease is the pollution in lake and mud [4]. Most people got infection through contacting with polluted water, food contaminated with urine, and soil from animals infected [5]. Leptospira bacteria was consist two type, first type
named pathogenic having the opportunity to disease, second type named saprophytic but did not consider as disease [6] [7]. Approximately Leptospira looks like morphology which did not differentiate whether the pathogenic and the saprophytic genus, but based on culture structure, antigenic or genetic nature method for these purposes [8].
The polymerase chain reaction (PCR) test could be taken for the diagnosis of leptospirosis in the biological laboratory by sampling the bacteria from blood, urine, tissues, and serum [9][10] while this test used the enzyme-linked immunosorbent assay (ELISA) to complete the diagnostic pathogenic Leptospira specimen [11][12]. The polymerase chain reaction (PCR) method needed a specific instrument, enough laboratory space, and also high expertise personnel. The microscopic agglutination test (MAT) defined a test which marked out antibodies in the serum of a patient by mixing together with Leptospira[13] [4]. Samples of polluted water and soil could be isolated and experimented for growth of pathogenic Leptospira, they take few weeks or months to complete the test [14] [7].
In recent years, researchers investigated several detection methods which contained optical, electrochemical, thermal and mass method, achieving a high accuracy of used biosensor is required to detect and monitor the microbial in polluted water. The used biosensor was contained a bio identify, bio transducer, and an electronic circuit which includes voltage, current, signal measurement, amplifier, and processor. The Polymers were widely used in biosensor electrochemistry applications because of their high conductivity that was compatible with molecular biology, stability environment and basic monomer synthesis [15]. Conductive polymers offer a range of monomers for innovative materials which allow new advances in the biosensor monitoring system [10]. Conducting polymers used by various applications in electrochemical biosensors for monitoring the environment for detection bacteria in the water. Polyvinyl alcohol (PVA) became popular because its wide range of potential invocation in optical, pharmaceutical, medical, microelectronics, advanced material and membrane field.
PVA is an important water-soluble polymer and extensively used in industries cause its inspired chemical and physical properties, non-toxicity, attractive chemical resistance, excellent film establishment potential and high crystal structure [16][17][18].
Polyaniline (PANI) had become a significant conducting polymer due to morphological and electrical properties [11]. PANI had a greatest advantage because it is rather inexpensive. Furthermore, it had a doping process perform the sensor appropriate for interacting microbes to the thin film surface. The interactions between a bacteria with surfaces of the Polyaniline (PANI) could produce a part of the signal in the current–
voltage (I-V) and electrochemical impedance spectroscopy (EIS) characteristic [12]
[13]. The conductivity biosensor can flow from the dopant to the polymer, the subsequent interaction between polyaniline and metal had been the response a wide range of applications [14]. The used metal alloy: Ferum (Fe), Aluminum (Al), Nickel (Ni), Manganese (Mn) by adding various ratios which doped with the polymers, it has increased this particular area of materials in terms of sensitivity and accuracy [15].
Polyaniline (PANI) based metal alloys expected high sensitivity, stability, quick response time and suitable with target bacteria. The criteria of Polyaniline (PANI) metal alloy thin films had been analyzed by UV-Vis spectroscopy. In this paper, the nanocomposite thin films of PANI-Fe0.5-Mn0.5, PANI-Fe0.5-Al0.5 and PANI-Fe0.5- Ni0.5 nanocomposites thin films were synthesized and fabricated by sol-gel method using the spin-coating technique. Compositions of the Fe-Al, Fe-Mn and Fe-Ni were investigated for achieving the high sensitivity of the used biosensor. The prototype of PANI-Fe0.5-Mn0.5, PANI-Fe0.5-Al0.5 and PANI- Fe0.5-Ni0.5 nanocomposites thin films based had been developed and performed using (IV) and (EIS) measurement to detect pathogenic of Leptospira bacteria in the water environment.
EXPERIMENTAL
Reagents and Chemicals
The precursors of iron nitrate (Fe(NO3)3), nickel nitrate (Ni(NO3)2), aluminum nitrate (NO3)3, manganese nitrate (Mn(NO3)2), polyvinyl alcohol (PVA), and aniline (C6H7N) were used as starting material. Bacteria of Benjo-Iso9 species Leptospira kmetyi [16] were provided by the Department of Veterinary Pathology & Microbiology, Faculty of Veterinary, University Putra Malaysia.
Preparation of substrate PANI-Fe0.5-Mn0.5, PANI-Fe0.5-Al0.5 and PANI-Fe0.5-Ni0.5 nanocomposites thin films
The preparation of the thin film substrates was done in three steps, firstly the polyvinyl alcohol (PVA) were dissolved in deionized water (DI) 30ml and produced transparent liquid. Secondly, the solution had been doped between a various ratio of metal with PANI-Fe0.5-Mn0.5, PANI-Fe0.5-Al0.5 and PANI-Fe0.5-Ni0.5 nanocomposites thin films, and lastly adding the aniline (1.25ml) into the solution at the temperature between 80°C and 90°C.
Fabrication of Biosensors
The biosensors had been synthesized by sol-gel method. Furthermore, the PANI-Fe0.5- Mn0.5, PANI-Fe0.5-Al0.5 and PANI-Fe0.5-Ni0.5 nanocomposites thin films had been deposited on glass substrates by using the spin coater technique brand Laurell Technologies Corporation model WS-400BX with a speed 2000 rpm in time period 20 sec. The thin films were fabricated on the glass substrate brand Corning, the size of thin films is 20 mm x 25 mm with the separation interval three combs. The thin film was annealed in a furnace at the temperature of 270 °C for 24 hours. The comb three intervals were sputtered silver was ejected off the ion with output 50W- 500W on the glass substrate of thickness 1000Ǻ thickness by using RF magnetron sputtering equipment. The copper wires were soldered to the silver electrode terminal for connecting between the measuring device brand GAMRY-Physical Electrochemistry and PANI-Fe0.5-Mn0.5, PANI-Fe0.5- Al0.5 and PANI-Fe0.5-Ni0.5 nanocomposites thin films. For safety purposes, all materials, tubes, plates, needles, pipettes and other
items using in the experiment should be soaked with a bleach solution during minimum 1 hour.
Current-Voltage (I-V) and Impedance Measurement for Sensitivity Performance
Current- Voltage (I-V) and Electrochemical impedance spectroscopy (EIS) were measured impedance by sweeping the frequency from 0 V- 5 V and 1 Hz to 50 kHz using GAMRY-Physical Electrochemistry instrument to determine the sensitivity of PANI- Fe0.5-Mn0.5, PANI-Fe0.5-Al0.5 and PANI-Fe0.5-Ni0.5 nanocomposites thin films to detection of Leptospira bacteria. The measurement system was started measuring whenever the biosensor was immersed once in the antiseptic water, and another in septic water which contained the concentrations 108 colony forming units (CFU/mL) of Leptospira Bacteria.
RESULTS AND DISCUSSION
The preparation and characterization of these nanocomposite thin films are discussed.
The formation of PANI-Fe0.5-Mn0.5, PANI-Fe0.5-Al0.5 and PANI-Fe0.5-Ni0.5 nanocomposites thin films were characterized by UV-vis spectroscopy, current-voltage (IV) measurements, and electrochemical impedance spectroscopy (EIS) characteristics.
UV-vis Spectra
The UV-vis was recorded by a PERKIN ELMER/Lambda 35 UV-Vis Spectrophotometer. UV–vis absorption was used to analyze the characteristics of both PANI-Fe0.5- Mn0.5, PANI-Fe0.5-Al0.5 and PANI-Fe0.5-Ni0.5 nanocomposites thin film to prove the UV-vis absorption spectra of ferum, aluminum, manganese and nickel in the structure of thin films shown in Figure 1. The absorption spectra of the spectrum of thin films, recorded in the wavelength range from 300 nm to 800 nm. The peak at 330 nm for iron doped with PANI-Fe0.5-Mn0.5, PANI-Fe0.5-Al0.5 and PANI-Fe0.5-Ni0.5 nanocomposites. UV-Vis-NIR spectroscopy has confirmed that the reactions in the thin film samples indicate the presence of metal transition [13].
Figure 1: Absorption spectra of UV–Visible spectra PANI-Fe0.5-Mn0.5, PANI-Fe0.5-Al0.5
I-V measurements
A current–voltage (I-V) characteristic is a relationship represented of PANI- Fe0.5- Mn0.5, PANI-Fe0.5-Al0.5, and PANI-Fe0.5-Ni0.5 nanocomposites thin films were determined the Leptospira bacteria. From the I-V measurements, the result has shown the difference of results between DI water and water with Leptospira bacteria as demonstrated in following Figure 1, Figure 2, Figure 3 and Figure 4.
Figure 2: 𝐼-𝑉 measurement of PANI-Fe0.5-Ni0.5 nanocomposites thin film sensor with Leptospira bacteria
Figure 3: 𝐼-𝑉 measurement of PANI-Fe0.5-Mn0.5 nanocomposites thin film sensor with Leptospira bacteria
Figure 4: 𝐼-𝑉 measurement of PANI-Fe0.5-Al0.5 nanocomposites thin film sensor with Leptospira bacteria
The result indicated that the interaction between the initial bodies of Leptospira those of gram- negative bacteria [19]. The reaction between surfaces molecules and microbe interactions could be observed that shown on the graph, it is depending on the ratio of PANI-Fe0.5-Mn0.5, PANI-Fe0.5-Al0.5, and PANI-Fe0.5-Ni0.5 nanocomposites thin films. All thin films were being tested with the Leptospira bacteria and had been good respond to the microorganism and perform a signal to the GAMRY measurement.
Figure 5: Sensitivity (S) of PANI-Fe0.5-Mn0.5, PANI-Fe0.5-Al0.5 and PANI-Fe0.5- Ni0.5 nanocomposites thin film sensor with Leptospira bacteria
The sensitivity of a biosensor was explained, the ratio of the response volume on imposing to the bacteria (Ie) with without impose to the bacteria (Io). Figure 5 shown the sensitivity (S) of Leptospira bacteria detection with PANI-Fe0.5-Mn0.5 PANI- Fe0.5-Al0.5 and PANI-Fe0.5-Ni0.5 nanocomposites thin films sensor with Leptospira bacteria who estimated using the formula below.
Where S is the sensitivity of biosensor terminal on Leptospira, Ie is the current when the biosensor was imposed to Leptospira, and Io is the current when the biosensors terminal was not imposed to Leptospira. Figure 5 shows the trend of maximum sensitivity that caused effects and impacts of PANI-Fe0.5-Mn0.5, PANI-Fe0.5-Al0.5, and PANI-Fe0.5- Ni0.5. Finally, the samples could be detected, and the variation of results was based on the type of following metals: ferum with nickel, aluminium and manganese [20].
Electrochemical Impedance Spectroscopy (EIS) Characteristics.
Electrochemical impedance spectroscopy is measured using a small excitation signal for characterizing changes at the surface thin film, this process depends on a diffusion of the surface which has a particular character of Leptospira bacteria[21]. The Nyquist impedance plots of the d PANI-Fe0.5-Mn0.5, PANI-Fe0.5-Al0.5, and PANI-Fe0.5- Ni0.5 nanocomposites are shown in Figure 6, Figure 7 and Figure 8, when the thin films were immersed into Leptospira bacteria samples. The impedance plots exchange at different points on the graph pattern that the cell wall gram-negative of the Leptospira bacteria, according to an effect on the thin film surface.
Figure 6: Nyquist impedance plot of PANI-Fe0.5-Ni0.5 nanocomposites thin film sensor with Leptospira bacteria
Figure 7: Nyquist impedance plot of PANI-Fe0.5-Mn0.5 nanocomposites thin film sensor with Leptospira bacteria
While the alternating sinusoidal potential was used, the impedance (Z) of the experimental measurement was a function of its resistance (Rs), capacitance (Cdl) and the applied frequency (f), as indicated in the equation below. Generally, the impedance between two components was inversely proportional with Rs (charged molecules) and unlike the case with Cdl (ionic of concentrations, type of ions and surface of properties) following formula below.
The estimated value of used impedance could be presented as the absenteeism. It was developed due the changes in number, expansion and morphological behavior of cell wall bacteria. When the cells were enclosed to the electrode terminal, the electron moves among electrodes, while a rising trend of the electron resistance area, and appearing situation of the signal. [22].
Figure 8: Nyquist impedance plot of PANI-Fe0.5-Al0.5 nanocomposites thin film sensor with Leptospira bacteria
CONCLUSION
In this paper, PANI-Fe0.5-Mn0.5, PANI-Fe0.5-Al0.5 and PANI-Fe0.5-Ni0.5 nanocomposites thin films had been synthesized and fabricated by the sol-gel method, the detection of Leptospira bacteria based on polymerization process. Based on the result, UV-Vis an absorption spectra which has been confirmed the formation of Fe, Mn, Al, and Ni nanoparticles in the PANI thin films nanocomposites structure. The prototype of biosensor had been applied to detect the Leptospira bacteria in water environments. This was proved by I-V and EIS graph according to two conditions named, deionized (DI) water without bacteria and water with Leptospira bacteria.
Various compositions metals were used with thin films to accurate the sensitivity.
ACKNOWLEDGMENTS
The research was funded by the Exploratory Research Grants Scheme (ERGS/1/2012/STG05/UKM/02/5), Photonic Technology Laboratory, Department of Electrical, Electronic and System Engineering, Universiti Kebangsaan Malaysia, Bangi, Selangor, Malaysia and Department of Veterinary Pathology & Microbiology, Faculty of Veterinary Medicine Universiti Putra Malaysia, Serdang, Selangor, Malaysia are gratefully acknowledged.
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