Development of an Enzymatic Biosensor Using Glutamate Oxidase on Organic–Inorganic-Structured, Electrospun Nanofiber-Modified Electrodes for Monosodium Glutamate Detection

Herein, dendrimer-modified montmorillonite (Mt)-decorated poly-Ɛ-caprolactone (PCL) and chitosan (CHIT)-based nanofibers were prepared. Mt was modified with a poly(amidoamine) generation 1 (PAMAMG1) dendrimer, and the obtained PAMAMG1–Mt was incorporated into the PCL–CHIT nanofiber’s structure. The PCL–CHIT/PAMAMG1–Mt nanofibers were conjugated with glutamate oxidase (GluOx) to design a bio-based detection system for monosodium glutamate (MSG). PAMAMG1–Mt was added to the PCL–CHIT backbone to provide a multipoint binding side to immobilize GluOx via covalent bonds. After the characterization of PCL–CHIT/PAMAMG1–Mt/GluOx, it was calibrated for MSG. The linear ranges were determined from 0.025 to 0.25 mM MSG using PCL–CHIT/Mt/GluOx and from 0.0025 to 0.175 mM MSG using PCL–CHIT/PAMAMG1–Mt/GluOx (with a detection limit of 7.019 µM for PCL–CHIT/Mt/GluOx and 1.045 µM for PCL–CHIT/PAMAMG1–Mt/GluOx). Finally, PCL–CHIT/PAMAMG1–Mt/GluOx was applied to analyze MSG content in tomato soup without interfering with the sample matrix, giving a recovery percentage of 103.125%. Hence, the nanofiber modification with dendrimer-intercalated Mt and GluOx conjugation onto the formed nanocomposite structures was performed, and the PCL–CHIT/PAMAMG1–Mt/GluOx system was successfully developed for MSG detection.


Introduction
Glutamate (Glu) is an amino acid in protein-containing foods [1]. Numerous studies have discovered that Glu is present in the cerebral cortex in one of the major intracellular signal pathways. Changes in the Glu concentration cause Huntington's disease [2,3]. Moreover, Glu is a crucial indicator for various other illnesses [4], such as musculoskeletal pain [5], tumors [6], and Alzheimer's disease [7]. Currently, Glu has been detected using multiple neurochemical probes, including carbon fiber microsensors based on enzymes or microdialysis [8]. Enzymatic biosensors utilize glutamate oxidase (GluOx) and glutamate dehydrogenase (GDH) as recognition components to detect Glu. Monosodium glutamate (MSG), a form of Glu, is a commonly used food additive that increases food's palatability [9], and it is hazardous. MSG releases neurotransmitters crucial to healthy physiological and pathological processes by acting on Glu receptors [10]. Excessive ingestion of MSG can cause health problems such as headaches, stomachaches, and neuronal excitotoxicity [11]. Thus, detecting MSG content in food is important to identify whether its amount exceeds permissible limits [12]. Therefore, developing reliable, fast, and specific methods for MSG detection is critical. Numerous electrochemical MSG biosensors have been created so far. For example, Devi et al. developed a novel immunosensor using gold nanoparticles

Modification of Mt with PAMAM G1 Dendrimer
Montmorillonite (Mt) was modified with PAMAM G1 via the cation exchange process. During this process, Na + ions between the Mt interlayers were exchanged with the quaternary alkyl ammonium ions in the PAMAM G1 . For this purpose, 0.5 g of Mt clay mineral was dispersed in 200 mL deionized water at room temperature overnight [29,30]. Simultaneously, 50 mL of a 0.02 mmol PAMAM G1 solution and an equivalent amount in relation to the cation exchange capacity of Mt was prepared in a different beaker and stirred. An aqueous, 1.0 M HCl solution was added to adjust the pH to 2.0-3.0 [29,30]. After stirring for a few hours, the protonated PAMAM G1 solution was slowly added to the neat Mt dispersion and left at room temperature overnight. The obtained PAMAM G1 -modified clay (PAMAM G1 -Mt) was precipitated using ultracentrifugation (at 18,000 rpm for 15 min). PAMAM G1 -Mt was washed with distilled water at least three times and filtered until no bromide ions were detected using an aqueous silver nitrate (AgNO 3 ) solution [29,30]. The resulting sample was dried in a vacuum at 35 • C.

Preparation of the PCL-CHIT/PAMAM G1 -Mt/GluOx Biosensors
A 10% (w/v) polycaprolactone (PCL) solution and a 0.5% (w/v) chitosan solution were separately prepared from 3:7 (v/v) formic acid to acetone solutions. PCL and CHIT solutions were mixed in a ratio of 7:3 (v/v), then stirred overnight at room temperature [29,30]. The solutions were filled into 2.0-mL syringes fitted with an 8.8 mm inner diameter metallic needle. The distance between the clean GCE, which was attached to the collector plate, and the syringe, which was fixed horizontally in a syringe pump (NE-300; New Era Pump Systems, Inc., New York, NY, USA) (tip-to-collector distance), was 18-19 cm in the electrospinning unit. The applied voltage and flow rate of the polymer solution were adjusted to~20 kV and~1.28 mL/h, respectively. After the electrode was covered with PCL-CHIT nanofibers, it was dried at 40 • C for 1 h. Then, 0.5% (w/v) Mt or PAMAM G1 -Mt was first added to a 7:3 (v/v) PCL-CHIT solution to prepare PCL-CHIT/Mt and PCL-CHIT/PAMAM G1 -Mt solutions, respectively [29,30]. The homogeneous solution was mixed overnight at room temperature and then poured into the syringes. The distance between the clean GCE and the syringe was 17-19 cm in the electrospinning unit. The applied voltage and flow rate of the polymer solution were~19 kV and~0.6 mL/h, respectively. Electrodes coated with bead-free PCL-CHIT/Mt and PCL-CHIT/PAMAM G1 -Mt nanofibers were dried at 40 • C for 1 h. Then, 10 µL of GluOx (0.2 U) enzyme was immobilized onto these electrodes using 2.5 µL glutaraldehyde (GA, 1.5%) as a crosslinking agent [17,18,31,40,43]. Afterward, the electrode was dried in an oven at 25 • C for 15 min. These modified PCL-CHIT/Mt/GluOx and PCL-CHIT/PAMAM G1 -Mt/GluOx were utilized in electrochemical measurements.

Electrochemical Measurements
All amperometric measurements were performed in a 10 mL electrochemical working medium containing 50 mM, pH 6.5 sodium phosphate buffer at −0.7 V versus an Ag/AgCl electrode at room temperature, and responses were recorded in µA. For the surface characterization of bare GCE, GCE/PCL-CHIT/PAMAM G1 -Mt, and GCE/PCL-CHIT/PAMAM G1 -Mt/GluOx, CV, DPV, and EIS measurements were carried out in 50 mM, pH 6.5 sodium phosphate buffer containing 5.0 mM potassium hexacyanoferrate (K 3 [Fe(CN) 6 ]) and 0.1 M KCl buffer. The CV and DPV measurements of bare GCE, PCL-CHIT/PAMAM G1 -Mt, and PCL-CHIT/PAMAM G1 -Mt/GluOx-coated GCE were conducted at potential rates of −0.4 to +0.8 V and −0.1 to +0.5 V with the scan rates of 50 and 25 mV/s, respectively. The EIS measurements of these GCEs were performed with frequencies in the range of 0.21 × 10 −4 to 10 kHz and an excitation voltage of 0.18 V, and superimposed on a dc potential of 0.01 V in the same electrochemical working medium.

Characterization of PAMAM G1 -Mt
As a 2:1 layered silicate, montmorillonite minerals consist of an octahedral layer between two tetrahedral layers. There are exchangeable cations between the Mt interlayers, which can easily be replaced by other cations, such as quaternary ammonium cations coming into the clay's environment via the cation exchange reactions. This method is performed to make hydrophilic silicate surfaces organophilic and to increase the layer spacing of the clay mineral [29,30]. The surface of Mt needs to be more hydrophobic to obtain the nanoscale dispersed Mt layers within the polymer matrix. For this purpose, Mt was modified with PAMAM G1, which contained quaternary alkyl ammonium salt, and was characterized using the FTIR, XRD, and TGA-DTG methods.
Firstly, the structural characterization of Mt and PAMAM G1 -Mt was performed using FTIR ( Figure 1A). From the FTIR spectrum of pure Mt, the band that appeared at~3635 cm −1 is attributed to the relative humidity in the Mt clay structure. The characteristic O-H stretching vibration of water between the layers appeared at~3430 cm stretches. After modification with PAMAM, the characteristic PAMAM G1 bands were also observed in the FTIR spectrum with Mt bands. The new bands were observed at 3080 and 2960 cm −1 owing to the -CH stretching vibrations in the PAMAM G1 structure. The strong band of -CH 2 bending vibrations is assigned to 1465 cm −1 ; the bands of PAMAM G1 N-H amine groups are designated to~1560 cm −1 .

Formation of PCL-CHIT/PAMAMG1-Mt and GluOx Conjugation on ESNFs
In the SEM images of the PCL-CHIT nanofibers grown with Mt and PAMAMG1 (Figure 2A-C), the incorporation of clays decreased the diameter of the ESNFs. Figure 2D shows the SEM images of PCL-CHIT/PAMAMG1-Mt/GluOx. After the conjugation of GluOx on the PCL-CHIT/PAMAMG1-Mt, the morphology of nanofibers changed to a sticky form because glutaraldehyde created covalent bonds between the amine groups of GluOx and PCL-CHIT/PAMAMG1-Mt ESNFs. The structural characterization of PCL-CHIT nanofiber was performed using FTIR analysis ( Figure 2E). Furthermore, the neat PCL and CHIT molecules were characterized via FTIR for their comparison with the nanofiber. According to the FTIR spectrum of neat CHIT, a strong, broad band in the region of 3354-3281 cm −1 corresponds to O-H and N-H stretching, namely, the intramolecular hydrogen bonds. The absorption band observed at ~2870 cm −1 can be attributed to C-H asymmetric stretching. These bands are the characteristics of polysaccharide molecules. The bands observed at ~1641 and 1560 cm −1 are attributed to C=O stretching and N-H bending, respectively. The C-H and O-H bending were confirmed using the bands that appeared at ~1414 and 1370 cm −1 , respectively. The absorption band located at 1153 cm −1 relates to the asymmetric stretching of the C-O-C bridge. The bands observed at 1062 and 1026 cm −1 correspond to C-O stretching. All FTIR spectral bands of chitosan correspond with those in the literature [45,46]. The FTIR spectrum of neat PCL was also investigated, and the PCL absorption bands located at ~2941 and 2864 cm −1 were assigned to asymmetric and symmetric -CH2-stretching, respectively. The band observed at 1722 cm −1 represents the stretching vibration of the carbonyl group in PCL. Symmetric and asymmetric C-O-C stretching were observed as strong bands at ~1240 and 1163 cm −1 [47]. The similarity of the FTIR spectrum of the PCL-CHIT nanofiber with that of neat PCL is remarkable. Most CHIT bands coincided with neat PCL bands. Therefore, all CHIT bands were not observed, owing to the considerably lower percentage of chitosan and interference with the The changes in the basal spacing (d001) between the layers after exchanging sodium ions with the PAMAM G1 were detected using XRD analysis. The XRD patterns of Mt and PAMAM G1 -Mt are presented in Figure 1B. The 2θ angle of Mt between layers was determined as 2θ = 7.65 • , indicating the regular repeats of silicate layers in the range of 3 • -9 • . The basal spacing (d001) of Mt was calculated as 11.54 Å using the Bragg equation corresponding to the diffraction angle. After the intercalation of PAMAM G1 with Mt, the diffraction angle shifted to the lower values of 2θ = 6.11 • in the same range. The basal spacing of PAMAM G1 -Mt was expanded to 11.54 Å from 14.45 Å after modifying Mt with PAMAM G1 . The increase in the basal spacing values indicates that the PAMAM G1 intercalates into the interlayer space of Mt. The interlayer distance increases with the alkyl chain length of the organic molecules. This result is comparable with our previous study on PAMAM with a variable alkyl chain length [29,30]. The basal spacing of the interlayers increases with the chain length of the PAMAM.
The thermal stability of Mt and PAMAM G1 -Mt is presented with TG/-DTG curves in Figure 1C,D. From the TGA thermogram, Mt showed~7.5% weight loss at 600 • C owing to the presence of volatile substances. In the DTG analysis of pure Mt, the degradation consists of two stages. The first weight loss was owing to the water adsorbed on the surfaces of the sheets at 0-120 • C. The second one arose from the loss of the adsorbed water in the inner parts of the layers owing to the dehydroxylation of the aluminosilicate lattices of the Mt structure at 635 • C [29]. The degradation of PAMAM G1 -Mt occurred in three steps. In addition to these peaks, the degradation of PAMAM on the surface and between the Mt layers was observed at~260 • C. After the modification, the degradation peak of Mt shifted toward the lower temperature (from 635 • C to 470 • C) compatible with the literature [44]. The amount of the organic cation content was determined to be 22.50% from the TG thermogram of the PAMAM G1 -Mt, owing to the adsorption of PAMAM on the surface and between the clay layers.

Formation of PCL-CHIT/PAMAM G1 -Mt and GluOx Conjugation on ESNFs
In the SEM images of the PCL-CHIT nanofibers grown with Mt and PAMAM G1 (Figure 2A-C), the incorporation of clays decreased the diameter of the ESNFs. Figure 2D shows the SEM images of PCL-CHIT/PAMAM G1 -Mt/GluOx. After the conjugation of GluOx on the PCL-CHIT/PAMAM G1 -Mt, the morphology of nanofibers changed to a sticky form because glutaraldehyde created covalent bonds between the amine groups of GluOx and PCL-CHIT/PAMAM G1 -Mt ESNFs. The structural characterization of PCL-CHIT nanofiber was performed using FTIR analysis ( Figure 2E). Furthermore, the neat PCL and CHIT molecules were characterized via FTIR for their comparison with the nanofiber. According to the FTIR spectrum of neat CHIT, a strong, broad band in the region of 3354-3281 cm −1 corresponds to O-H and N-H stretching, namely, the intramolecular hydrogen bonds. The absorption band observed at~2870 cm −1 can be attributed to C-H asymmetric stretching. These bands are the characteristics of polysaccharide molecules. The bands observed at~1641 and 1560 cm −1 are attributed to C=O stretching and N-H bending, respectively. The C-H and O-H bending were confirmed using the bands that appeared at~1414 and 1370 cm −1 , respectively. The absorption band located at 1153 cm −1 relates to the asymmetric stretching of the C-O-C bridge. The bands observed at 1062 and 1026 cm −1 correspond to C-O stretching. All FTIR spectral bands of chitosan correspond with those in the literature [45,46]. The FTIR spectrum of neat PCL was also investigated, and the PCL absorption bands located at~2941 and 2864 cm −1 were assigned to asymmetric and symmetric -CH 2 -stretching, respectively. The band observed at 1722 cm −1 represents the stretching vibration of the carbonyl group in PCL. Symmetric and asymmetric C-O-C stretching were observed as strong bands at~1240 and 1163 cm −1 [47]. The similarity of the FTIR spectrum of the PCL-CHIT nanofiber with that of neat PCL is remarkable. Most CHIT bands coincided with neat PCL bands. Therefore, all CHIT bands were not observed, owing to the considerably lower percentage of chitosan and interference with the PCL bands. The band observed at~1371 cm −1 (O-H bending) proves the existence of CHIT in the nanofiber. SEM images were marked (100 different points) to evaluate the distributions of the nanofiber diameters using ImageJ. Figure 3 shows the histograms of the diameter distributions of PCL-CHIT, PCL-CHIT/Mt, PCL-CHIT/PAMAM G1 -Mt, and PCL-CHIT/PAMAM G1 -Mt/GluOx. The calculated diameter distributions of PCL-CHIT, PCL-CHIT/Mt, PCL-CHIT/PAMAM G1 -Mt, and PCL-CHIT/PAMAM G1 -Mt/GluOx were found as 356.61 ± 12.89, 141.33 ± 4.41, 227.15 ± 5.55, and 332.26 ± 10.73 nm, respectively. The addition of Mt to PCL-CHIT nanofibers decreased their size. As clays contain cations, they increase the conductivity of the solution [48]. The intercalation of Mt with PAMAM G1 and the decoration of PCL-CHIT nanofibers with PAMAM G1 -Mt increased the nanofiber's diameter. There was a decrease in conductance with the addition of organoclay, so the diameter of PCL-CHIT/PAMAM G1 -Mt nanofibers increased [49]. Due to the swelling properties of CHIT and Mt, the diameters of PCL-CHIT/PAMAM G1 -Mt nanofibers increase after GluOx immobilization in an aqueous solution [50,51].
PCL is a biodegradable polyester with the chemical formula (C 6 H 10 O 2 ) n , and chitosan (C 6 H 11 NO 4 ) n is a copolymer of N-acetyl-D-glucosamine and D-glucosamine. According to the SEM-EDS results of the PCL-CHIT nanofibers, carbon, oxygen, and nitrogen elements were observed ( Figure 4A). By adding Mt (Al 2 H 2 O 12 Si 4 ) to the polymer solution, aluminum and silicon elements were seen in the PCL-CHIT/Mt nanofibers ( Figure 4B). After the modification of Mt with the PAMAM G1 dendrimer, the percentage of nitrogen in PCL-CHIT/PAMAM G1 -Mt also increased with the nitrogen in the PAMAM G1 structure ([NH 2 (CH 2 ) 2 NH 2 ]:(G = 1); dendri PAMAM(NH 2 ) 8 ) ( Figure 4C). GluOx contains a riboflavin nucleic acid derivative (flavin adenine dinucleotide (FAD)) because of its flavoprotein struc-ture. Nucleotides are bound via phosphodiester bonds in FAD [40]. Therefore, phosphorus was observed in PCL-CHIT/PAMAM G1 -Mt/GluOx ESNFs after GluOx immobilization ( Figure 4D). This indicated immobilization was successfully performed.
PCL-CHIT/PAMAMG1-Mt, and PCL-CHIT/PAMAMG1-Mt/GluOx were found as 356.61 ± 12.89, 141.33 ± 4.41, 227.15 ± 5.55, and 332.26 ± 10.73 nm, respectively. The addition of Mt to PCL-CHIT nanofibers decreased their size. As clays contain cations, they increase the conductivity of the solution [48]. The intercalation of Mt with PAMAMG1 and the decoration of PCL-CHIT nanofibers with PAMAMG1-Mt increased the nanofiber's diameter. There was a decrease in conductance with the addition of organoclay, so the diameter of PCL-CHIT/PAMAMG1-Mt nanofibers increased [49]. Due to the swelling properties of CHIT and Mt, the diameters of PCL-CHIT/PAMAMG1-Mt nanofibers increase after GluOx immobilization in an aqueous solution [50,51].   PCL is a biodegradable polyester with the chemical formula (C6H10O2)n, and chitosan (C6H11NO4)n is a copolymer of N-acetyl-D-glucosamine and D-glucosamine. According to the SEM-EDS results of the PCL-CHIT nanofibers, carbon, oxygen, and nitrogen elements were observed ( Figure 4A). By adding Mt (Al2H2O12Si4) to the polymer solution, aluminum and silicon elements were seen in the PCL-CHIT/Mt nanofibers ( Figure 4B). After the modification of Mt with the PAMAMG1 dendrimer, the percentage of nitrogen in PCL-CHIT/PAMAMG1-Mt also increased with the nitrogen in the PAMAMG1 structure ([NH2(CH2)2NH2]:(G=1); dendri PAMAM(NH2)8) ( Figure 4C). GluOx contains a riboflavin nucleic acid derivative (flavin adenine dinucleotide (FAD)) because of its flavoprotein structure. Nucleotides are bound via phosphodiester bonds in FAD [40]. Therefore, phosphorus was observed in PCL-CHIT/PAMAMG1-Mt/GluOx ESNFs after GluOx immobilization ( Figure 4D). This indicated immobilization was successfully performed.   The electrochemical surface of the developed biosensor was characterized using CV, DPV, and EIS. K 3 [Fe(CN) 6 ] was used as a redox probe during electrochemical measurements. According to cyclic voltammograms, peak currents were calculated as 43.194, 31.831, and 28.113 µA for bare, PCL-CHIT/PAMAM G1 -Mt, and PCL-CHIT/PAMAM G1 -Mt/GluOx-modified GCEs, respectively. Redox peak potential separations were 0.082, 0.116, and 0.186 mV for bare, PCL-CHIT/PAMAM G1 -Mt, and PCL-CHIT/PAMAM G1 -Mt/GluOx-modified GCEs, respectively. As shown in Figure 5A, the current responses decreased after each modification owing to the limitation of the K 3 [Fe(CN) 6 ] transfer to the electrode surface. As the CV results, the peak currents gradually decreased as the electrode surfaces were covered with ESNFs. They were calculated as 92.750, 46.108, and 21.864 µA for bare, PCL-CHIT/PAMAM G1 -Mt, and PCL-CHIT/PAMAM G1 -Mt/GluOx-modified GCEs, respectively ( Figure 5B). For the characterization of biocatalytic transformations on modified electrode surfaces, EIS is a commonly used efficient electrochemical technique. EIS gives information about the capacitance and load transfer resistance of the modified electrode's surface. The charge transfer resistance (Rct) of K 3 [Fe(CN) 6 ] was calculated using the diameters of the semicircles created in the Nyquist plots of the EIS spectra for the modified GC electrodes. When the electrode surface was modified, the load-transfer transition became more difficult, and the diameter of the semicircle increased.

PCL-CHIT/PAMAMG1-Mt/GluOx for MSG Detection
The first optimization step of the working medium trials was the determination of

PCL-CHIT/PAMAM G1 -Mt/GluOx for MSG Detection
The first optimization step of the working medium trials was the determination of optimum pH. The effects of pH on the biosensors' responses were analyzed using PCL-CHIT/Mt/GluOx and PCL-CHIT/PAMAM G1 -Mt/GluOx biosensors in 50 mM sodium phosphate buffer (from pH 6.0 to 8.0) while adding 0.25 mM MSG into the working cell as a substrate. Thus, the effect of PAMAM G1 on the nanofiber structure was tested to compare two developed biosensors. The amperometric biosensor's responses were observed in µA and calculated relatively. As a result of the measurements, the optimum pH values of the PCL-CHIT/Mt/GluOx and PCL-CHIT/PAMAM G1 -Mt/GluOx biosensors were 7.5 and 6.5 in a sodium phosphate buffer, respectively. As shown in Figure 6, the pH value shifted from an alkaline to an acidic region. The presence of amino groups in the PAMAM G1 dendrimer could be considered why the optimum pH of the PCL-CHIT/PAMAM G1 -Mt/GluOx biosensor was more acidic [29,40].  As a result of the enzymatic catalysis reaction of GluOx, the current was changed over time. The current variations after the addition of MSG are displayed in Figure 7A. Both developed biosensor system responses decreased at 0.5 mM of MSG ( Figure 7B). The linear ranges were determined to be from 0.025 to 0.25 mM of MSG as a substrate using the equation y = 4.953x − 0.080 (R 2 = 0.974) for the PCL-CHIT/Mt/GluOx biosensor and from 0.0025 to 0.175 mM MSG using the equation y = 5.423x − 0.023 (R 2 = 0.985) for the PCL-CHIT/PAMAM G1 -Mt/GluOx biosensor (with a limit of detection of 7.019 µM for PCL-CHIT/Mt/GluOx and 1.045 µM for GCE/PCL-CHIT/PAMAM G1 -Mt/GluOx [n:8]) ( Figure 7C). The sensitivities were 4.953 and 5.423 µA mM −1 for PCL-CHIT/Mt/GluOx, and PCL-CHIT/PAMAMG 1 -Mt/GluOx, respectively. This way, lower MSG concentrations could be detected using this biosensor system developed by modifying Mt with PAMAM G1 . Due to PAMAM G1 -modified Mt, the immobilization process was more successful at increasing the distances between the layers of clays in the PCL-CHIT/PAMAM G1 -Mt/GluOx biosensor. Table 1   ChBD-GluOX/PB/SPC CV 25 µmol/L to 300 µmol/L 9.0 µmol/L Fermentation broth samples [54] GluOx/PMPD/Pt modified GRE CV 2.0-550 µM 0.536 µM Cucumber juice and fruit [55] PtNP decorated 10-110

OR PEER REVIEW
Vegetable soup, soy sauce, stock cube, One of the most important characterization studies of biosensors is repeatability trials. For an ideal biosensor system, almost identical results are expected to be obtained in consecutive measurements under the same conditions with the same electrode. The lower standard deviation (SD) and coefficient of variation (cv) indicate the reproducible biosensor system. For this purpose, five consecutive measurements were recorded with 0.0175 mM MSG using the developed biosensor system. According to these measured values, SD and CV were calculated as ±0.0016 and 5.585%, respectively. Herein, immobilizing the GluOx enzyme onto the nanofiber-coated electrode surface with glutaraldehyde as a crosscovalent binding agent provides high repeatability. Furthermore, for the operational stability determination of the PCL-CHIT/PAMAM G1 -Mt/GluOx biosensor, amperometric measurements were recorded every 0.5 h for 6 h using 0.0175 mM MSG as a substrate under the same conditions. No significant decrease in activity was observed for the first 5 h, though the biosensor's activity decreased by 63.415% at the end of 6 h.
To determine the substrate specificity of PCL-CHIT/PAMAM G1 -Mt/GluOx, measurements were taken using aspartic acid, lysine, and glycine as substrates, and the results were relatively comparable ( Figure 8A). To examine whether the PCL-CHIT/PAMAM G1 -Mt/GluOx biosensor was open to interference, measurements were performed by adding ascorbic acid, 3-acetamidophenol, glucose, and uric acid at the same concentrations to 0.025 mM MSG in the working buffer. The biosensor response to MSG was assumed to be 100%, and the responses to other components were compared relatively [59]. The results show no significant interference effect of the other compounds ( Figure 8B).

PCL-CHIT/PAMAM G1 -Mt/GluOx for MSG Detection in Real Samples
To test the applicability of the PCL-CHIT/PAMAM G1 -Mt/GluOx biosensor to real samples, MSG determination was performed in tomato soup. Firstly, 22 g powdered soup was dissolved in 170 mL water and centrifuged for 15 min at 7000 rpm. Then, this supernatant was diluted 250 times with water, and a few drops of concentrated HCl were added to the solution. Amperometric measurements were taken using the prepared MSG added soup (by standard addition method) solution, and the biosensor's response followed. According to the equation y = 5.423x − 0.023 [R 2 = 0.985] for the PCL-CHIT/PAMAM G1 -Mt/GluOx biosensor, the concentrations of standard MSG solution and MSG-added soup were found to be 0.030 ± 0.0025 (mM ± SD) and 0.029 ± 0.006 (mM ± SD), respectively. In line with these findings, recovery was calculated as 103.125%, indicating the successful application of the developed biosensor system to real samples.

PCL-CHIT/PAMAMG1-Mt/GluOx for MSG Detection in Real Samples
To test the applicability of the PCL-CHIT/PAMAMG1-Mt/GluOx biosensor to real samples, MSG determination was performed in tomato soup. Firstly, 22 g powdered soup was dissolved in 170 mL water and centrifuged for 15 min at 7000 rpm. Then, this supernatant was diluted 250 times with water, and a few drops of concentrated HCl were added to the solution. Amperometric measurements were taken using the prepared MSG added soup (by standard addition method) solution, and the biosensor's response followed. According to the equation y = 5.423x − 0.023 [R 2 = 0.985] for the PCL-CHIT/PAMAMG1-Mt/GluOx biosensor, the concentrations of standard MSG solution and MSG-added soup were found to be 0.030 ± 0.0025 (mM ± SD) and 0.029 ± 0.006 (mM ± SD), respectively. In line with these findings, recovery was calculated as 103.125%, indicating the successful application of the developed biosensor system to real samples.

Conclusions
PCL-CHIT/PAMAMG1-Mt/GluOx enzymatic biosensor was prepared and tested for sensitive, specific, and fast detection of MSG in real samples. First, Mt was intercalated with PAMAM, and the obtained PAMAMG1-Mt was successfully incorporated with the PCL-CHIT structure. PCL-CHIT/PAMAMG1-Mt was an alternative matrix for the covalent conjugation of GluOx to prepare the MSG biosensor. The usability of biosensors in the food industry was studied. The PCL-CHIT/PAMAMG1-Mt/GluOx has good features, which can be integrated with point-of-care sensor technologies.

Conclusions
PCL-CHIT/PAMAM G1 -Mt/GluOx enzymatic biosensor was prepared and tested for sensitive, specific, and fast detection of MSG in real samples. First, Mt was intercalated with PAMAM, and the obtained PAMAM G1 -Mt was successfully incorporated with the PCL-CHIT structure. PCL-CHIT/PAMAM G1 -Mt was an alternative matrix for the covalent conjugation of GluOx to prepare the MSG biosensor. The usability of biosensors in the food industry was studied. The PCL-CHIT/PAMAM G1 -Mt/GluOx has good features, which can be integrated with point-of-care sensor technologies.