Characterization of indicator electrodes using the potentiometric biosensor method as urea sensor with XRD, SEM-EDS, UV-Vis and FTIR

The purpose of this study is the characteristics of the indicator electrode which has been coated with a membrane with XRD, SEM-EDS, UV-Vis and FTIR. Research has been carried out using XRD, SEM-EDS, UV-Vis and FTIR tests on immobilized indicator electrodes 0.0350 g PVA-Enzyme coated with PVC-KTpClPB 0.0120 g and 0.0500 g. The method used was the biosensor potentiometric method with the urease enzyme immobilization technique in PVA coated with PVC-KTpClPB. To see the difference in the absorbance spectrum pattern of each PVA-Enzyme composition coated with PVC-KTpClPB against wavelength with UV-Vis, PVA-Enzim solution as a reference and PVC-KTpClPB solution as the first analysis 0.0120 g and as the second analysis 0,0500 g. To see the PVA-enzyme membrane coated with PVC-KTpClPB in the two compositions above was characterized by FTIR and SEM-EDS. To see the indicator electrode that had been immobilized by PVA-enzyme coated with PVC-KTpClPB in the two compositions above, it was characterized by XRD. It was concluded that the best results were found on the immobilization indicator electrode of 0.0350 g PVA-Enzyme coated with 0.0350 g PVC-0.0500 g KTpClPB.


Introduction
Urea in the blood is transported to the kidneys from the liver filtered and excreted through urine [1] . Urea in milk contributes 55% of non-protein nitrogen. At normal levels of urea in natural milk are 3.0-6.0 mM, depending on the animal feed habits, the allowable limit for the concentration of urea in milk is usually ≤ 11 mM. In mammals urea is synthesized in the liver due to the end product of biochemical reactions involved in the formation of endogenous ammonia detoxification. Detection of urea through urea sensors is very much needed development, there are more rapid examples of capacitive multienzyme biosensor field effects based on comparative studies of Drop-Coating and Nano-Spotting Techniques [2]. Biomedical sensors used to detect metabolites, ions, and viruses are updated by [3] in the manufacture of electrochemical biosensors. Preparation of Urea Biosensor for Dynamic Liquid Measurement by [4]. A biosensor made in the form of a membrane is used for rapid detection [5]. The analysis used as follows (1) study of the membrane surface of the indicator electrode that has been modified is carried out by scanning electron microscopy (SEM) of membrane morphology [4; 6; 7], (2) FTIR spectral pattern shows immobilization of urease deposited on the electrodes [1; 8; 6; 7], (3) UV-Vis spectrum patterns [9]. (4) XRD diffraction spectrum patterns [7]. On the basis of the explanation above, the researcher performed the characterization of the electrode membrane using sequential analysis of XRD, UV-Vis, SEM-EDS and FTIR on two indicator electrodes with different compositions of KTpClPB, namely 0.0500 g and 0.0120 g.

Method
The indicator electrode was made using the biosensor potentiometric method with the urease enzyme immobilization technique on the PVA-Enzim membrane and then coated with PVC-KTpClPB. The PVA-Enzyme membrane was derived from a solution of 0.0350 g PVA and 1 mg urease enzyme, the PVC-KTpClPB membrane was derived from a PVC-KTpClPB solution [10;11]. The first method of making PVA solution consists of 0.0350 g of PVA dissolved in 10 mL of hot water to cool somewhere. The second method 0.0350 g PVC + 0.0500 g KTpClPB is dissolved in 10 mL THF in a glass tube and covered with a plastic / aluminum poil cover. The third method 0.0350 g PVC + 0.0120 g KTpClPB was dissolved in 10 mL THF in a glass tube and covered with a plastic / aluminum poil cover. The fourth method of 1 mg urease enzyme is dissolved in 0.5 mL of water mixed with alcohol in a ratio of 50% : 50%. The indicator electrode is made of tungsten wire with a diameter of 1 mm 99.99% Aldrech metal, amounting to three with a length of 1.5 cm.
The urease enzyme is dropped 1 drop in the PVA solution, stirred until blended to become PVAenzyme. The tungsten indicator electrode was immersed in a 0.0350 g PVA-Enzyme solution to immobilize the urease enzyme and coated with 0.0350 g PVC + 0.0120 g KTpClPB. There are three tungsten used which will be used for dyeing 1x, 2x, and 3x which are denoted E A-1x , E A-2x and E A-3x . Likewise, the tungsten indicator electrode was immersed in 0.0350 g PVA-Enzyme solution for immobilization of the urease enzyme and coated with PVC 0.0350 g + 0.0500 g KTpClPB, the indicator electrode was denoted E B-1x , E B-2x and E B-3x .
Next, the six electodes E A-1 x, E A-2x , E A-3x and E B-1x , E B-2x and E B-3x were analyzed with XRD Figure  1. Making the indicator electrode begins with analysis of 0.0350 g PVA-Enzyme solution, 0.0350 g + PVC solution KTpClPB 0.0120 g and 0.0350 g PVC solution + 0.0500 g KTpClPB using UV-Vis. UV-Vis analysis through 0.0350 g PVA-Enzyme solution as reference, 0.0350 g PVC solution + 0.0120 g KTpClPB as analysis 1 and 0.0350 g + KTpClPB 0.0500 g PVC solution resulted in the absorbance spectrum pattern of Figures 4a and 4b.
For the manufacture of PVC-KTpClPB coated PVA-Enzim membranes, one drop of PVA-Enzim solution is dropped on clean glass using a dropper on the glass plane in two different places. After drying, add another drop once to obtain two layers of PVA-Enzyme in each place. After drying the second drop of each PVA-Enzyme layer, one of which is a drop of 0.0120 g of PVC-KTpClPB solution and the other is a drop of 0.0500 g of PVC-KTpClPB until dry so that a PVA-Enzim membrane is coated with PVC-KTpClPB 0.0120 g denoted M 1 and PVA-Enzim coated with PVC-KTpClPB 0.0500 g in M 2 denotation. Each membrane M 1 and M 2 were analyzed by SEM-EDS see

Results and discussion
The six indicator electodes E A-1x , E A-2x , E A-3x and E B-1x , E B-2x and E B-3x were analyzed by XRD and obtained six XRD diffraction spectrum patterns, see Figure 1. The XRD diffraction spectrum pattern of the six indicator electrodes is at intensity between the 2 theta angle of 40-50 degrees. PVA hybrid nanofibers are located between an angle of 2 theta 30-40 degrees [12]. The nanostructures with CA and PVA doped ZnO composites with intensities between 2 theta angle of 40-50 degrees [13]. PVA / SiO 2 -TiO 2 is about 2 theta 20 degrees [16]. PVA between angle 2 angles 35-40 degrees [14]. PVA / PAA / Fe 3 O 4 between 2 angles 15-20 degrees [15]. The best results of the XRD spectrum pattern of 3 the two compositions were obtained at the indicator electrodes E A-2x and E B-2x . The best spectral pattern was selected on the E B-2x indicator electrode with immobilization of PVA-Enzim 2x coated with PVC-KTpClPB 0.0500 g 1x according to [10].  Based on Figure 1, the XRD diffraction spectrum pattern of the two best samples is the E A-2x and E B-2x indicator electrodes, the membranes of the electrodes were analyzed by SEM-EDS. Each membrane electrode indicator E A-2x and E B-2x is denoted by M 1 and M 2 . The results obtained from SEM-EDS analysis are the morphology and spectral patterns of the indicator electrode membranes E A-2x and E B-2x as shown in Figure 2 and Figure 3. SEM morpology was analyzed at observation diameter 50 μm, magnification of 1000 x operating voltage 20 kV.
There are differences in SEM morphology and the EDS spectrum pattern of the indicator electrodes E A-2x and E B-2x from the membranes M 1 and M 2 . Based on pore SEM morphology [4; 6] which is more clearly visible and brighter in Figure 2a for the indicator electrode E B-2x , namely the M 2 membrane. Likewise with the EDS spectrum pattern [10; 11; 13; 14], the highest peaks of the EDS spectrum pattern are between energies 1-3 keV. The elements contained in the next EDS spectrum pattern will be linked to FTIR analysis and the chemical structure of the substance used.  Based on the XRD diffraction spectrum pattern analysis of the indicator electrode, also the morphological analysis and EDS spectrum pattern of SEM of the indicator electrode membrane. The next step is to analyze the M 1 and M 2 indicator electrode membrane solutions using UV-Vis through the absorbance spectrum pattern of wavelengths. The analysis of the absorbance spectrum pattern was traced from a wavelength of 200-1000 nm. The analysis results obtained can be seen in Figure 4a of M 1 solution and Figure 4b of M 2 solution. Based on the spectral pattern, the highest absorbance peaks of M 1 and M 2 were 3.725 a.u and 7.246 a.u at wavelengths 315 nm and 291 nm. There was a shift in the absorbance peak of the M 1 solution and M 2 solution [12], as well as a change in the width of the absorbance peak which affected the detection range of the sensor [11].  The shift of the absorbance peak with respect to the wavelength of the M 1 and M 2 solutions was supported by [16] using PVA / SiO 2 -TiO 2 . According to [8; 9; 15] it is expected that there is no shift of the absorbance peak with respect to the wavelength, which is expected to be a symmetrical UV-Vis absorbance spectrum pattern [17]. Also by FTIR %transmittance spectrum pattern and XRD diffraction spectrum pattern with cerium oxide nano samples (CeO 2 NCs) as a function of urea concentration by [18].