Identification of Leptospira in water by Fe-Pd-doped polyaniline nanocomposite thin film

Leptospirosis disease was caused by rat urine which contains the genus Leptospira bacteria. In this study, the fabrication of Pd-Fe-doped polyaniline nanocomposite thin films for the determination of the genus Leptospira bacteria thin films has been investigated. Pd-Fe-doped polyaniline nanocomposite thin films were fabricated by sol–gel spin coating method. The electrode sensors were immersed in the Leptospira solution. The resulting materials were investigated using field-emission scanning electron microscopy, atomic force microscopy, transmission electron microscopy, and current–voltage measurement. The atomic force microscopy images show the specific morphology films’ structure for Leptospira detection, whereas the field-emission scanning electron microscopy image shows the irregularity of clump nanoparticles in thin film surfaces. Transmission electron microscopy result shows that metal alloy (Fe-Pd) embedded in the polymer matrix. Current–voltage measurement with and without incubation of the thin film into Leptospira solution was done to show the relationship between concentration bacteria versus current. The result shows that polyaniline-Fe0.4-Pd0.6 nanocomposite thin film has higher sensitivity in detecting Leptospira, where it has performed with the highest percentage of the sensitivity of 16.9%. Besides that, selectivity tests were conducted to distinguish the existence of Leptospira, Pseudomonas aeruginosa, and Staphylococcus aureus bacteria. These results confirm the potentials of polyaniline metal alloys’ nanocomposite thin films to be used for Leptospira bacteria detection in water.


Introduction
Leptospirosis is a zoonotic disease caused by the genus Leptospira that consists of saprophytic and pathogenic Leptospira. Pathogenic strain can be detrimental to both animals and humans. 1 These bacteria are carried by rodents or domestic animals, which are considered an immediate host. Animals and humans have known to be infected through direct contact with urine, polluted soil, and water sample. Leptospira bacteria are contagious in humid tropical countries because this is their method of adaptation. This disease has been reported to be threatening to various groups of people especially those working as veterinarians, farmers, abattoir workers, meat inspectors, and workers in rodent control sectors. 2 Bacterial detection of nanocomposites can be made possible by converting the electrochemical signal into a quantity of electrical response. The electrical signals generated through the biochemical reaction between the thin active surface and the target bacteria, can be quantified, and measured for a reliable bacterial detection method. Currently, the detection of these bacteria is extremely timeconsuming. For instance, the Microscopic Agglutination Test (MAT) needs 8 days or more to provide positive result in regards to the diagnosis of these bacteria. 3 Enzymelinked immunosorbent assay (ELISA) method is considered inadequate and needs time to stabilize for the detection of Leptospira. 4 Polymerase chain reaction (PCR) method uses both urine and clinical blood samples in a test which could lead to contamination. 5,6 Difficulties in detecting Leptospirosis occur at the initial stages of this disease because it needs specific instruments, specific and sensitive samples, a large laboratory area, and highly skilled experts. Due to the problems stated above, there is a rising interest in using nanocomposite thin film as a detector. We are fabricating metal alloys-polymer thin film lab-on-a chip for a portable Leptospira biosensor that may take 1-2 days of detection. Polyvinyl alcohol (PVA) can be used as stabilizers in the chemical synthesis for particle size control of polyaniline (PANI) where it acts as a cross-linking agent to enhance the properties of PANI. 7 PVA is preferred due to being a good material for metal nanoparticle production, high thermal stability, chemical resistance, electrical conductivity, water solubility, and high mechanical strength. 8 This is the main reason to incorporate metal nanoparticles into PVA where the concentration and distribution of metals can be controlled in the polymer matrix. The incorporation of metal alloys' nanoparticles into a polymer matrix will make this sensor much more effective and carries advantages in terms of usage in hazardous and remote places. This sensor can also be used in conditions where the number of bacteria varies constantly and thus needing highly sensitive and accurate detector. 9 Previous studies have utilized the use of metal oxide, such as ZnO and TiO 2 , which shows to exhibit anticorrosive and self-cleaning functionalities and was synthesized using a green chemistry approach. 10,11 PANI is a promising substitute for these materials as it has better conductivity, high cycle stability, and cleanliness, making it a reliable candidate to use as bacterial detection.
This study demonstrates the application of PANI-Fe-Pd nanocomposites thin films as the active sensing material for the detection of saprophytic and pathogenic Leptospira in the 10 8 CFU mL À1 in water. The morphological structures of the thin films were analyzed using atomic force microscopy (AFM) and field-emission scanning electron microscopy (FESEM). This study aims to help researchers detect Leptospira in contaminated waters without the inherent disadvantages of conventional detection methods such as MAT, ELISA, and PCR. To achieve that goal, this new material that incorporates metal alloys in PANI is designed to obtain fast and highly sensitive results for in situ Leptospira detection that can serve as an effective public preventive measure against Leptospirosis disease.

Synthesis of samples
About 2.5 g of PVA solution was added into 40 mL of deionized (DI) water and heated to 90 C. About 0.5 g of iron nitrate and palladium nitrate was added into the PVA solution following the formula of Fe (1Àx) -Pd (1Àx) with x representing the weight ratios of Fe and Pd (x ¼ 1, 0.8, 0.6, 0.4, 0.2, and 0.0). About 1.25 mL of aniline and 1.0 M of nitric acid (HNO 3 ) were added to each of these solutions. All the solutions were mixed at 90 C 24 h À1 until it changes to a dark brown color indicating that the solution has become PANI-Fe-Pd nanocomposite. The nanocomposite solution was spin-coated on glass substrates at the speed of 1800-2000 r min À1 for 18-20 s using a spin coater model WS-400BX from the brand Laurell Technologies Corporation, USA. The glass substrates were heated for 10-15 min after the spin coat. These processes were repeated for four to five times until the thin film was obtained. Finally, the thin films were annealed in a furnace at 250 C 24 h À1 .

Fabrication of the sensor
A comb structure of silver electrode was sputtered on the thin film to obtain a thickness of 1000 Å using RF magnetron sputtering equipment manufactured by T-M Vacuum Products, Inc. USA . Basically, dimensions of the used thin film size were 20 mm Â 25 mm, the measured width of a silver layer was 2.2 mm, and separations between three combs were 1.2 mm. Finally, a copper (Cu) wire was soldered onto the silver layer as a terminal electrode. It connects to the GAMRY-Physical Electrochemistry (GAMRY Instruments, USA) for the sensor's performance measurement as shown in Figure 1.

Preparation of bacteria samples
The Leptospira bacteria samples were categorized based on the concentration of 10 8 CFU mL À1 (colony forming units per milliliter). All the wet samples were obtained from the Department of Veterinary Pathology & Microbiology, Faculty of Veterinary, Universiti Putra Malaysia.

Sensor performance
The sensor performance was tested using GAMRY Physical Electrochemistry Instrument G-300 as shown in Figure 2. I-V measurement was recorded by the GAMRY instrument when the thin films were immersed in DI water and Leptospira solution. The thin films were exposed to Leptospira solution: 10 8 CFU mL À1 for 4-5 min. These measurements were repeated for five times. For safety purposes, all materials, tubes, plates, needles, pipettes, and other items used in the experiment should be soaked with a bleach solution for a minimum of 1 h.

Morphological studies
FESEM image in Figure 3 shows the distribution of nanoparticles ranging from 80 nm to 100 nm. The arrangement of the thin film presents an irregularity pattern where the metal alloys' nanoparticles are surrounded by polymer on the thin films' surfaces. 12 The particle showed that the thin film has agglomerated. 13 This condition appears after the addition of the doping aniline with PVA during the chemical oxidation process. 14,15 It also happens due to the characteristic of PVA. Besides that, by coating the metal alloys, the sensors become highly beneficial for electrochemical sensing applications. This platform increases the reaction between the bacteria and films' surface via absorption of the gram-negative charged microbes within the film structure. 9,16 TEM images in Figure 4 show the morphology of PANI-Fe-Pd nanocomposites particles. The metal alloys' (Fe and Pd) nanoparticles are in a spherical pattern with the diameter size of around 10-40 nm. Incorporation of the PANI and metal alloys (Fe and Pd) forms nanocomposites, and it is clearly embedded in the polymer matrix. Meanwhile, the metal alloys-coated PANI is encapsulated and agglomerated. 17,18   AFM image in Figure 5 shows the morphology of the thin films, which contains metal alloys (Fe and Pd) on the polymer substrates. The thin films' surfaces look smooth and homogeneous, which produces high sensitivity for Leptospira detection. 19 The AFM images show the incorporation of PANI-Fe-Pd on the film causing their morphology to bind to the Leptospira bacteria. 20,21 The average roughness and grain size of the thin film were obtained at the range of 2.138 nm and 43.72 nm, respectively.

Current-voltage and sensitivity measurement
The I-V curve shows the current that is flowing through a film when a voltage is applied to the electrode. These results can be used to differentiate which of the sensor is immersed in a bacteria solution or clean water. The sensors were immersed in pathogenic Leptospira solution in the 10 8 CFU mL À1 range. Figure 6 shows the current measured across the thin film that was exposed to DI water and Leptospira concentration range of 10 8 CFU mL À1 . PANI-Fe 0.4 -Pd 0.6 shows the highest curve compared to the others. An interaction between the thin films and Leptospira showed the presence of conductivity due to the incorporation of PANI and metal alloys. 22 The result indicated that the cell wall of Leptospira is gram-negative bacteria. 23 The corresponding calibration curve presents a linear relationship between the peak current and the value of Leptospira concentration. In brief, the PANI-     The efficiency of PANI-Fe-Pd nanocomposites thin film in detecting Leptospira is determined by tabulating the sensitivity (S), where the ratio of the response volume on imposing to bacteria I e is being compared to impose without bacteria I o , using the following formula 23 Figure 7 shows the sensitivity of PANI-Fe-Pd nanocomposites thin films toward the Leptospira concentration. It has been found that PANI-Fe 0.4 -Pd 0.6 nanocomposites thin film gave a higher value. The samples of PANI-Fe 0 . 4 -Pd 0.6 performed the highest percentage of sensitivity of 16.9%, whereas PANI-Fe 0.0 -Co 1.0 only performed 3.9% of sensitivity toward Leptospira bacteria. 24 These results show that the sensor can detect Leptospira for the concentration range of 10 8 CFU mL À1 . It is proven that high selectivity of the thin film is achieved by doping it with PANI metal alloys during the sensor preparation.

Selectivity test and error analysis
The selectivity test is used to determine other bacterial signals in comparison with Leptospira. In this test, we used both the Pseudomonas aeruginosa (gram-positive) bacteria and Staphylococcus aureus (gram negative) to see the tendency of thin films toward these bacteria. 25 Figure 8 shows the selectivity test of PANI-Fe 0.4 -Pd 0.6 curve against DI water, Leptospira, P. aeruginosa, and S. aureus bacteria. The current of P. aeruginosa and S. aureus bacteria slightly increase in association with DI water but are lower under Leptospira. The Leptospira linear regression equations are obtained, expressed as y ¼ 0.0009x À 0.0016 with a correlation coefficient of 0.9103. P. aeruginosa linear regression equations is expressed as y ¼ 0.0009x À 0.0016 with a correlation coefficient of 0.9103, whereas the S. aureus linear regression equations is expressed as y ¼ 0.0009x À 0.0016 with a correlation coefficient of 0.9103. These demonstrate the high ability of thin film to detect Leptospira bacteria in comparison to other bacteria. 26,27 Figure 9 shows the error bar of current-voltage measurements and Table 1 tabulates the error bars value for cycles of I-V measurements. The sensors are regarded as having high linearity if the error bars value obtained is still small after recording the measurement five times (15 s intervals with a total of 75 s). The value of the error bar changes around 5% after several measurements. The testing protocols are used to demonstrate the stability of these sensors. The standard deviation for each sensor was estimated under zero condition after calibration. Based on the above results, the sensors' linearity response to the environment is very stable. 28,29 Sensor performance error   analysis shows that the film sensor for Leptospira has high stability.

Conclusion
In this study, we successfully synthesized and fabricated PANI-Fe-Pd nanocomposite thin films via the sol-gel method to be utilized as a Leptospira microbial sensor. The metal alloys are successfully incorporated into the PANI matrix to increase the sensitivity of the thin film surface, making it extremely effective for Leptospira detection. In addition, the AFM images show the roughness and grain size of the film that could bind the cell of microbes. Besides that, the in-depth images of FESEM show irregularity of nanoparticles on the thin film with the deposition of metal alloy, which is beneficial for electrochemical sensing. TEM result shows that metal alloy (Fe-Pd) is embedded in the polymer matrix. The I-V measurement can be used to evaluate the sensitivity of each pathogenic Leptospira concentration. The samples of PANI-Fe 0 . 4 -Pd 0.6 gave the highest percentage of sensitivity where it about 16.9%. The lab-ona-chip method is used to detect Leptospira bacteria because this method is very fast and highly sensitive. A lab-on-achip biosensor is a compatible platform for the portable detection of Leptospira bacteria.