Application of Surface Plasmon Resonance Sensor with Polypyrrole Chitosan Graphene Oxide layer to Detect the Napropamide

A polypyrrole Chitosan Graphene Oxide (PPy-Chi/GO) composite layer was prepared using the electro-chemical method. The PPy-Chi/GO layer was used to measure the low concentration of napropamide. In this study, the PPy-Chi/GO composite layer was deposited on the gold layer side of a microscope’s glass slide in different thicknesses. The morphology, thickness, roughness, and refractive index of the layer were obtained using field effect scanning electron microscopy, profilemeter, atomic force microscopy, and the surface plasmon resonance technique. The structure of the composite layer was investigated using X-Ray diffraction. The peak that were observed from X-ray diffraction was at 9.9°, corresponding to GO, and the broad peak between 25° and 27° was related to polypyrrole and chitosan. The PPy-Chi/GO layer was used to measure the concentration of napropamide dissolved in hexane in the range of 0.01 to 100 ppm using the surface plasmon resonance technique; the limitation of sensor was 0.1 ppm.


Introduction
Napropamide is N, N-diethyl-2-(1-naphthyloxy) propionamide and a polar herbicide that is readily soluble in water, acetone, chlorobenzene, ethanol, xylene, hexane, and dichloromethane. The empirical formula of napropamid is C17H21NO2. The molecular weight and the specific gravity of napropamide were 271.4 g/ml and 0.584, respectively [1]. Napropamide is used to control broadleaf weeds and annual grasses on numerous agriculture products [2]. Numerous analytical methods are used to measure the amount of napropamide that is used in agriculture, food, and the environment. Gas Chromatography (GC) [3][4][5], High Performance Liquid Chromatography (HPLC) [6,7], and UV-visible spectroscopy [2] are the methods that commonly are used to detect and measure the concentration of napropamide in chemical and agricultural laboratories. The disadvantages of the mentioned methods including nonportability, chemical knowledge, standard sample, and the costs of device and the experiment.
The surface plasmon resonance (SPR) technique is a versatile and accurate method for the detection of biomolecules [8,9]. In order to enhance the sensitivity and selectivity of its, the gold layer should be improved with a sensing layer [8], such as Polypyrrole [10,11], Polypyrrole -chitosan [12,13], or Polypyrrole Multi-walled carbon nanotubes [14].
Polypyrrole (PPy) is a high-potential, conducting polymer that is derived from pyrrole. It has conjugated double bonds and good environmental stability and high conductivity [15]. PPy has been used as a biosensor [16,17] and a gas sensor [18,19]. The affinity of PPy for organic molecules is based on the intrinsic affinity of the PPy backbone, the affinity of the side groups, and binding to immobilized receptors [20].
Chitosan is poly (b-1-4)-2-amino-2-deoxy-D-glucopyranose, which is derived from chitin. Chitin is a component in the exoskeleton of shellfish. Chitosan has affinity to binding with heavy metal and biomolecules, because the amino (-NH2) and hydroxyl groups are abundant functional groups on chitosan [21,22], and in acidic medium, the chitosan's amino groups have positive charges; hence, the chitosan can interact with some biomolecules that have negative charges [23]. The major factor to evaluate the interaction of the chitosan with biomolecules and ions is the degree of deacetylation [24] in the chitosan.
Graphene Oxide (GO) is derived from graphite oxide crystals and it is a single-atomic-layered of all-sp 2 hybridized carbon. GO has application in solar cells [25], medicine, biology [26][27][28], and inorganic optoelectronic devices [29]. The molecular structure of GO includes the hydroxyl (OH − ), epoxy (-COO -) groups, and the carboxyl groups, (-COO − ) are the main functional group of GO at the molecular structure [30,31].
In this contribution, the polypyrrole chitosan/Graphene oxide (PPy-Chi/GO) composite layer (sensing layer) was prepared using the electro-chemical method to measure the concentration of napropamide using the surface plasmon resonance technique. The field effect electron microscopy FE-SEM) and X-ray diffraction (XRD) were used to characterize the sensing layer.

Preparation of Graphene Oxide
In 2011, Huang et al., [32] reported the preparation of graphene oxide. Briefly, the graphene oxide was obtained from oxidation of graphite. In this process, H2SO4:H3PO4 (320:80 ml), graphite splinter, and KMnO4 (18 g) were mixed using a magnetic stirrer for 3 days to form the GO; the color of the mixture changed to dark brown. Afterward, the H2O2 solution was added to stop the oxidation process. The graphite oxide that formed was washed three times with 1 M of HCl aqueous solution and repeatedly with deionized water until a pH of 4-5 was achieved. The washing process was carried out using a simple decantation of supernatant via a centrifugation technique having a centrifugation force of 10,000 g. During the washing process using deionized water, the graphite oxide experienced exfoliation, which resulted in at thickening of the graphene solution, forming a GO gel. The final concentration of GO was 2 mg/ml, and for this experiment, the final solution was dissolved in deionized water, achieving 0.3 mg/ml.

Preparation of sensing layer
The gold layer was deposited on microscope glass slide using sputtering coating device (K757 Turbo) at a thickness of 45 nm prior to electro-deposition of sensing layer. The preparation of a sensing layer has been described in ref. [12]. Briefly, the sensing layer was coated on a gold layer by electrochemical deposition using a potentiostat (Model PS 605). The anodic potential of the working electrode was 1.1V relative to a saturated calomel electrode. The polymers were potentiostatically prepared in a solution containing 0.3M pyrrole, 0.1M PTS dopant, and 0.7% w/v of CHI in acetic acid at room temperature, and the concentration of GO was 1 mg/L in final solvent. The morphology and structure of prepared layer on gold side of microscope glass slide were achieved X-Ray diffraction (XRD, Ital Structure, APD 2000) and field effect scanning electron microscopy (FE-SEM, FEI NANOSEM 230). Figure 1 is the SPR setup based on prism in the Kretschmann configuration. It contains a He-Ne laser, a chopper, a polarizer, a pinhole, a precision rotation stage, a high index (ZF52, Foctek), a flow cell, a silicon detector, and a lock-in amplifier. The lock-in amplifier was connected to a computer to register the SPR sensor. The PPy-Chi/GO composite layer (sensing layer) was attached to a high index prism using refractive index matching gel, and the flow cell was attached to the prism. The probe samples flowed into the flow cell separately. The rotation stage was rotated up to 40° in increments of 0.016°. The intensity of the laser beam was registered when the rotation stage was stopped momentarily. The computer program saved the angle of rotation and the intensity of He-Ne laser. The analysis of SPR signals were done based on the Fresnel equation. The transverse mode (TM) was generated using a polarizer for excitation of the surface plasmon. The reflectivity (R=rr * ) [12] was calculated from the reflective coefficient (r) of the layers based on the matrix method [33]. The resonance angle was obtained with minimizing the equation

Preparation of solution
In order to prepare the solution of napropamide, 100 mg of C17H21NO2 was dissolved in 1 liter of hexane at 35°C for producing in 100 ppm. Then, other concentrations (0.1 ppm, 0.5 ppm, 1 ppm, 5 ppm, 10 ppm, 25 ppm, 50 ppm, and 75ppm) were prepared by a systematic dilution of the 100 ppm of C17H21NO2 in solution.  Figures 2(a) and 2(b) show the FE-SEM image for the PPy-Chi layer [12,13,[34][35][36] and the PPy-Chi/GO composite layer. The PPy-Chi appeared within the GO sheet, which was covered by the PPy-Chi composite.  Figure 3 shows the XRD pattern of PPy-Chi/GO. The broad peak about the 25° to 27° is related to PPy-Chi [37] which is match to literature. The peak rise at 9.9° is correspond to GO. This results authenticated the PPy-Chi composite file was formed with graphene oxide (GO).  The electro-deposition time (t) was shifted from 1to 30 min to determine a different thickness layer. The profilemeter measured the thickness of the layers with a limitation of about 5 nm. The thickness of the PPy-GO composite layers was increased from 8 to 60 nm, and Fig. 4 shows the variation of thickness versus electro-deposition time (t). The refractive indices of the layers were measured using the surface plasmon resonance technique. The layers were attached to the prism separately, and they were in contact with water (n=1.3317). Afterward, the SPR signal was registered (Fig. 5) for analysis and calculation of the refractive index of the layer. The resonance angle and the refractive index of each layer were calculated using the matrix method based on the Fresnel equation [9,12,33]. Figure 5 shows that the resonance angle was shifted from 54.783° to 70.079° and that it was a function of the refractive index and the thickness (d) of the layers. The real (n) and imaginary (k) parts of the refractive index were shifted from 1.7872 to 1.7504 and 0.164 to 0.218, respectively. Figure 6 depicts that the imaginary part of the refractive index increased monotonically as expected from the Kramers-Kroning formula [38]. The pertinent parameters are listed in Table 1.

Sensing the napropamide
The pure hexane (n= 1.3724) was loaded in the flow cell, and it contacted to the PPy-Chi/GO sensing layer with 18 nm thickness. The SPR signal was registered to determine the baseline prior to doing the experiments. Figure 7 shows the SPR signal at the baseline, and the angle of resonance was 61.662°. The napropamide was dissolved in hexane prior to the experiment at concentrations in the range of 0.1 to 100 ppm. The samples were loaded in the flow cell separately, and the SPR signals were registered using a computer program. In order to obtain the sensogram, the experiment was repeated 15 times at room temperature for each concentration of napropamide. The SPR signals were analyzed using the matrix method, and the resonance angles were obtained using Eq. (1). The angle shifts were calculated by subtracting the angle of resonance for each concentration at different times from the angle of resonance at the baseline. Figure 8 shows the sensograms for different concentrations of napropamide. The sensograms indicated the variation of angle shift with time. As a result, variation of the angle shift for 0.5 ppm was larger than for 0.1, and the variation of angle shift was constant after 250 minutes, which was its terminal value.
where   and t are the angle shift and response time of the sensor, respectively. The angles shifts at the terminal value are a function of the concentration of napropamaide. Figure 9 shows the variation of the angle shift at the terminal value versus concentration of napropamaide. The data fit well to Langmuir equation [39] well, as follows: where max   , C , and K are the maximum value of resonance angle shift, concentration of ions, and affinity constant, respectively.

Conclusion
A polypyrrole-chitosan/ graphene oxide layers were prepared using electro-deposition in the range of 8 to 60 nm thickness The XRD pattern of PPy-Chi/GO layer confirmed the formation of PPy-Chi in the presence of GO. The refractive indices of the layers were measured using the SPR technique. Consequently, the real and imaginary parts of refractive index were in the range of 1.7872 to 1.7504 and 0.164 to 0.218, respectively. Napropamide was dissolved in hexane with different concentration in the range of 0.1 to 100 ppm. PPy-CHi/GO sensing layer could detected the napropamide and the angle shift was in the range of 0.012° to 2.003°. Consequently, PPy-Chi/GO sensing layer can detect the napropamide with the limitation about 0.1ppm.