Biodegradable film based on cellulose nanofiber/methylene blue/vitamin C: investigation of physicochemical properties and usability as a kit for identification of oxidants (hydrogen peroxide)

Biodegradable film based on cellulose nanofiber/methylene blue/vitamin C: investigation of physicochemical properties and usability as a kit for identification of oxidants (hydrogen peroxide) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Biodegradable film based on cellulose nanofiber/methylene blue/vitamin C: investigation of physicochemical properties and usability as a kit for identification of oxidants (hydrogen peroxide) Sina Sadeghi, Sajad Pirsa, Narmela Asefi, Mehdi Gharekhani This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5274857/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted 13 You are reading this latest preprint version Abstract In this study, cellulose nanofiber film was modified with methylene blue pigment and vitamin C (Cel/MB/VC). The physicochemical, structural, thermal resistance, and antibacterial characteristics of the prepared films were investigated using SEM, FTIR, XRD, TGA, etc. The rate of absorption and release of methylene blue from the prepared film was studied. Films containing methylene blue and vitamin C were used as a kit to detect hydrogen peroxide. The obtained results showed that methylene blue and vitamin C increased the thickness of the film and the elongation of the film. Both methylene blue and vitamin C agents reduced moisture content and water vapor permeability. Both methylene blue and vitamin C significantly increased the antioxidant and antimicrobial properties (against Escherichia coli and Staphylococcus aureus ) of the film. SEM images showed that the diameter of cellulose fibers is between 20 and 100 nm, which methylene blue and vitamin C completely cover their surface and the surface porosity of the film. FTIR spectra confirmed the electrostatic interactions between cellulose, methylene blue and vitamin C. According to the XRD results, cellulose has a crystalline structure, which vitamin C improved this crystalline property. According to TGA results, methylene blue and vitamin C caused more thermal stability of cellulose film. The release of methylene blue from the film was reported to be 15%. In the presence of hydrogen peroxide, the color of the films containing methylene blue and vitamin C changed from white to blue. Films containing methylene blue and vitamin C at the same time showed good performance as a hydrogen peroxide detection sensor (kit). The highest sensitivity of the sensor for measuring hydrogen peroxide was 0.299 (100 mg/ml) with a detection limit of 1.9 (100 mg/ml). The prepared film has the ability to be used in smart packaging of foods sensitive to oxidation such as oils. Biological sciences/Biochemistry Health sciences/Biomarkers Physical sciences/Chemistry Biodegradable film Kit oxidation/regeneration Antioxidant Antibacterial Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction The release of synthetic and petroleum polymers into the environment causes environmental pollution and creates many biological problems for humans and animals. Due to the fact that these polymers are not easily decomposed, the use of these polymers in various industries, including food packaging, faces many problems. Therefore, in recent years, the use of biodegradable polymers in food packaging has become very popular. Biodegradable polymers of plant and biological origin can be a good substitute for petroleum polymers. While these polymers have suitable physical and chemical properties for food packaging, they are easily decomposed when released in the environment and do not have environmental pollution problems (Pirsa and Mohammadi, 2021 ; Colnik et al., 2020 ; Pirsa et al., 2023 ). Biopolymers with protein, polysaccharide and lipid structures or their composites are used to produce biodegradable plastics. Cellulose and its derivatives are of special importance among different biological sources for the preparation of biodegradable films. Because cellulose is found in abundance in nature and is relatively cheap and available, and its structure can be modified easily (Liu et al., 2021 ; Yorghanlu et al., 2022 ; Farooq et al., 2020 ; Erfani et al., 2023 ). Cellulose is an organic compound that is known as the most abundant biopolymer on earth. Cellulose is a complex carbohydrate or polysaccharide consisting of hundreds to thousands of glucose molecules that are linked together and form a chain. Unlike animals, plants, algae and some bacteria and other organisms have the ability to produce this substance. Cellulose is the main structural molecule in the cell walls of plants and algae. Cellulose has many important derivatives. Most of them are biodegradable and renewable resources. Cellulose-derived compounds are usually considered non-toxic and non-allergenic. Derivatives include cellulose nanofiber, celluloid, cellulose acetate, methylcellulose, hydroxypropylmethylcellulose and carboxymethylcellulose, all of which are used in the preparation of biodegradable films (Gupta et al., 2019 ; Reis et al., 2019 ). Methylene blue is a functional dye that acts as a red-ox indicator and has different colors (blue/colorless) in oxidized or reduced states. One of the tests to determine the quality of milk, with which the number of bacteria in raw milk is estimated, is the methylene blue regeneration test. Chemically, methylene blue is a crystalline substance and a type of organic chloride salt, which is included among thiazine dyes. Thiazine dye means that it has oxazine or antioxidant properties (Seroglazova et al., 2022 ). Methylene blue is often used in textile industries, aquariums, medical, dental and chemical laboratories. Another property of methylene blue is the ability to protect the heart and vascular system (Evora et al., 2015 ). One of the most important uses of methylene blue is its use as a disinfectant in aquariums. Aquariums are often infected with fungi and infectious bacteria, which endangers not only the health of the fish, but also the eggs of the fish. In these cases, by adding methylene blue to the aquarium water, fungal infections and bacteria are eliminated (Lin et al., 2015 ; Perni et al., 2010 ). Vitamin C is an antioxidant (or regenerative agent) that has the ability to prevent damage from free radicals and dangerous chemicals such as cigarette smoke. Free radicals play a role in the occurrence of some diseases, including cancer, heart disease and arthritis. This vitamin protects the skin from the harmful effects of the ultraviolet rays of the sun. Also, this vitamin helps to increase the immunity of the body and the strength of gums and teeth. This vitamin also makes collagen (the strongest part of connective tissue that holds all body parts together) and is effective in preventing blood cholesterol from rising and blood clots in the veins (Pehlivan, 2017 ; Chiarappa et al., 2018 ). Active and smart packaging are among the new packaging methods that have attracted the attention of many scientists (Abdolsattari et al., 2020 ; Shabkhiz et al., 2021 ). Kits (color sensors) sensitive to the concentration of carbon dioxide, labels sensitive to the amount of oxygen, time-temperature labels and labels sensitive to changes in the pH of food are among the kits that are recently used in the smart packaging of food products (Abdolsattari et al., 2022 ). Packages containing smart kits, due to having special identifiers, can detect environmental conditions and changes in food and provide consumers with information about the quality and whether the food is healthy or unhealthy by means of these indicators. Smart food packaging does not work directly to increase the shelf life of food, but rather provides information about food quality to stakeholders in food supply chains (Mustafa and Andreescu, 2018 ). In this research, an active and intelligent biodegradable film was designed that, in addition to being able to delay the oxidation of food and increase its shelf life, at the same time, it can intelligently monitor the oxidation process in food products and increase its shelf life and estimate its expiration date. For this purpose, cellulose nanofiber film modified with methylene blue and vitamin C was used to prepare smart film. Methylene blue has a blue color in its natural state, which becomes colorless in the composite with vitamin C (as a reducing agent). Therefore, cellulose film modified with methylene blue and vitamin C is a white film. This film has the ability to detect the oxidizing agent. In this research, hydrogen peroxide was used as a soft oxidant to investigate the sensing behavior of the prepared cellulose film. Using mathematical equations, linear relationships were established between the color changes of the sensor and the concentration of hydrogen peroxide, and it was used for qualitative and quantitative measurement of hydrogen peroxide. The obtained results showed that the cellulose film modified with methylene blue and vitamin C has suitable physicochemical properties to be used as a smart marker in food packaging and can identify and measure hydrogen peroxide as a smart marker. 2. Materials and methods 2.1. Chemicals Cellulose film with Nano fibrous structure (thickness 30 to 100 nm) and porosity 5 to 10 micrometers (molecular weight 100,000 to 200,000 Daltons) was obtained from Zardab Tabriz (Iran). Hydrogen peroxide 35%, methylene blue, vitamin C, sodium hydroxide, silica gel and other chemical compounds used were obtained from Merck (Germany) and Aldrich (USA) companies and were used without further purification. 2.2. Preparation of cellulose/methylene blue/vitamin C film To prepare standard methylene blue solution, 500 mg of commercial methylene blue powder was dissolved in 1 liter of distilled water. Dissolution was continued at room temperature while stirring for 10 minutes. In order to prepare films, first, cellulose films were prepared in dimensions of 10×10 cm. In a 100 ml beaker, 50 ml of distilled water was added and some methylene blue solution was added to it at three levels (0, 50 and 100 µL - according to the Table 1 -A) and dissolved. Next, the cellulose film was immersed in the prepared solution for 10 minutes. After 10 minutes, the blue film was removed from the solution and dried in an oven at 50°C. Then the colored film was immersed in 50 ml of 5% NaOH solution for 10 minutes. Then the film was removed from the solution and dried again in the oven at 50°C. The dried film was immersed in a solution containing vitamin C at three levels (0, 2.5 and 5% - according to the Table 1 -A) for 10 minutes. At this stage, the color of the film turned white. At the end, the film was removed from the solution and dried in an oven at 50°C (Fig. 1 -a). The dried films were stored in black plastic bags at refrigerator temperature until the tests were performed. 2.3. Physical and chemical characteristics of the prepared films 2.3.1. Thickness The thickness of each film was measured at 5 different points. The obtained numbers were averaged and reported. The thickness of the film was used to determine mechanical resistance and water resistance, etc. In this test, a digital micrometer (Insize Digital Outside Micrometer 3108-25A) was used. 2.3.2. Mechanical characteristics The mechanical properties of the films include elongation at breaking point (EAB) and tensile strength (TS) are important factors for food packaging. To check the mechanical properties, the samples were conditioned for 24 hours in special conditions. Special conditions included relative humidity RH = 55%. The films were cut with a special cutter in dimensions of 1×8 cm in a dumbbell shape. The films were placed between the two jaws of the device, and the initial distance between the two jaws is 50 mm. The upper jaw was moved relative to the lower jaw at a speed of 5 mm/min. The mechanical properties were recorded by a computer. A Texture analyzer-brand histometer (Zwick/Roell model FR010, Germany) was used to perform this test. 2.3.3. Moisture content To measure the moisture of the films, first the films was cut into 3×3 cm dimensions. Then, the cut films were kept in a desiccator for 24 hours in specific humidity conditions (55% relative humidity) at a temperature of 25°C. The desiccator contained silica gel. At this stage, the weight of the films was measured and recorded as the initial weight. Then, in order to completely remove the moisture from the films, the film sample was heated at 100°C for 2 hours and finally its final weight was measured and recorded. The following equation was used to calculate the moisture content of the film. Humidity (%) = \(\frac{{\varvec{W}}_{\varvec{i}}-{\varvec{W}}_{\varvec{f}}}{{\varvec{W}}_{\varvec{i}}}\) ×100 (1) Wi: Initial film weight Wf: Final film weight 2.3.4. Water vapor permeability (WVP) Permeability relative to water vapor was measured by gravimetric method. For this test, the test film was sealed in a glass vial with a height of 4 cm containing silica gel to maintain zero percent relative humidity (RH 0%) in the vial. The vials had an inner diameter of 6.4 cm and their outer diameter was 8.9 cm. Also, the exposed surface of the vials was 26 cm 2 . The vials were placed in a controlled temperature (38 ± 1°C) and relative humidity (90 ± 3%). The transfer of water vapor was determined from the increase in the weight of the vial. Changes in cell weight were recorded as a function of time. The slope of weight changes versus time (after reaching steady state) was calculated by linear regression. Water vapor transmission rate (WVTR) was defined as the slope (g/d) divided by the transfer area (m 2 ). After penetration tests, WVP was calculated as follows (Khwaldia, 2013 ): \(\:WVP=\frac{WVTR\times\:X}{\varDelta\:p}\) [g µm/m 2 /d/ kPa] (2) Where X is the thickness of the paper and Δp is the partial pressure difference of water vapor throughout the film and its numerical value is 9.5 kPa. 2.3.5. Antioxidant property DPPH (2,2-diphenyl-1-picrylhydrazy) radical quenching method was used to measure the antioxidant power of the films. For this test, to prepare film extract, 25 mg of each film was dissolved in 4 ml of distilled water for 2 minutes. Then, 2.8 ml of film extract solution was mixed with 1 mM methanolic DPPH solution (0.2 ml). The obtained solution was stirred with a vortex 2000 rpm for 2 minutes and kept in a dark place for 1 hour. The absorbance of the solution was recorded using a spectrophotometer (Model T60 UV, USA) at a wavelength of 517 nm. The following equation was used to calculate the antioxidant percentage. $$\text{Antioxidant activity (%)}=\frac{{A}_{b}-{A}_{s}}{{A}_{b}}$$ 3 A b : Absorption rate of the control sample (DPPH methanolic solutions: 1 mM) A s : Sample absorption rate 2.3.6. Antibacterial activity Agar diffusion method was used to determine the antibacterial property of the films. For this purpose, the films were cut into discs (with a diameter of 15 mm) and then placed on Mueller Hinton agar plates containing Staphylococcus aureus ATCC6538 and Escherichia Coli ATCC13706, with a concentration of (10 7 CFU/mL). took The plates were incubated for 24 hours at 37°C. After 24 hours, the radius of bacterial non-growth halos around the films (in millimeters) was measured with a precision caliper. 2.3.7. Scanning electron microscopy (SEM) The technique of scanning electron microscope (ZEISS, SIGMA, Germany) was used to investigate the polymer structure, surface characteristics, porosity and particle distribution of the prepared films. In this method, the surface of the film samples was covered with a thin layer of gold before analysis. The accelerator voltage of 20 kV was used for the adjustment device. Imaging of the surface of the samples was done with different magnifications after placing the films in the special position of the sample in the device. 2.3.8. Fourier Transform Infrared Spectroscopy (FTIR) FTIR device (Tensor 27, Bruker, Germany) was used to investigate physical or chemical interactions between film components. To perform this test, first the films was dried and completely powdered. The obtained powder was mixed with KBr at a ratio of 1 to 20. The powder mixture was turned into a thin film with a special pressing machine. FTIR spectra of each sample were recorded separately. The spectrum of the samples was recorded with a resolution of 4 cm -1 in the range of 400–4000 cm -1 . 2.3.9. X-ray diffraction (XRD) XRD technique was used with the help of X-ray diffractometer (Kristalloflex D500, Siemens, Germany) to check the crystalline or amorphous structure. In this technique, first, the film samples were placed in the special position of the device (sample cell). The primary rays were irradiated to the sample at ambient temperature and the reflected rays were collected in the range of angle 2θ = 0–80°. The XRD spectra of the samples were drawn automatically by the machine. Cu Kα radiation source was used at a wavelength of 0.154 nm. The specifications of the device include X-ray generator at 40 kV and 40 mA. 2.3.10. Thermogravimetric analysis (TGA) To check the thermal stability of the films, the TGA test was used by a thermal analyzer (Linseis – L81A1750, Germany). For this purpose, films were prepared in the form of 10 mg samples. The film samples were heated under a nitrogen atmosphere of 50 cm 3 /min in aluminum cups in the temperature range of 30–600°C. The heating rate was 10°C/min. The empty aluminum cup was considered as a reference and the TGA curve was drawn and recorded by the device. 2.4. Absorption and release of methylene blue Considering that the UV-Vis spectrum of methylene blue has maximum absorption at three wavelengths of 250, 300 and 665 nm, and the maximum absorption is at the wavelength of 665 nm, the wavelength of 665 nm was used to investigate the absorption and release of methylene blue. To check the absorption of methylene blue, the UV-Vis spectrum of the solutions used to prepare the film was measured before and after the absorption of methylene blue by the film, and the amount of absorption was calculated from the decrease in the intensity of the absorption peak at the wavelength of 665 nm (Eq. 4). To check the release rate, the films containing methylene blue were immersed in 100 ml of distilled water and after 24 hours, the absorbance of the solution was measured at the wavelength of 665, and the release rate of methylene blue was checked from Eq. 5. \(\:Absorption\:\left(\%\right)=\frac{{A}_{\text{M}\text{B}1}-{A}_{\text{M}\text{B}2}}{{A}_{\text{M}\text{B}1}}\) ×100 (4) \(\:Release\:\left(\%\right)=\frac{{A}_{\text{M}\text{B}\text{i}}-{A}_{\text{M}\text{B}\text{r}}}{{A}_{\text{M}\text{B}\text{i}}}\) ×100 (5) In this regard, A MB1 is the absorption rate of methylene blue solution before coating on cellulose film and A MB2 is the absorption rate of methylene blue solution after coating on cellulose film. Also, A MBi is the absorption of the initial solution (standard) of methylene blue, and A MBr is the absorption of the released solution of methylene blue. 2.5. Cel/MB/VC film sensing behavior 2.5.1. Color characteristics of the sensor In this step, the color factors of the used films were analyzed. Considering that the film changed from white to blue, factor b was further investigated. In this factor, negative numbers indicate the intensity of the blue color, and as the number moves towards more positive numbers, the blue color decreases. To record color features, the system designed in previous studies was used (Fig. 1 -c) (Alizadeh et al., 2023 ). 2.5.2. Calibrating the sensor and sensing H 2 O 2 Hydrogen peroxide was used as a soft oxidant to calibrate the sensor and check the response of the sensor to oxidizing agents. Based on this, different concentrations of hydrogen peroxide were prepared and placed in contact with the sensor, and the response of the sensor was calculated with the Eq. 6 . Finally, the calibration curve of the sensor was drawn as the response of the sensor to different concentrations of hydrogen peroxide and the figures of merit of the sensor were calculated. $$\:Response=\frac{{b}_{1}-{b}_{0}}{{b}_{0}}\times\:100$$ 6 In this regard, b 0 is the value of b factor before contact with hydrogen peroxide and b 1 is the value of b factor after contact with hydrogen peroxide. 2.6. Statistical analysis The statistical analysis and analysis of the data obtained in this research was done in three parts. In order to examine the effect of two factors, the amount of methylene blue and vitamin C on the physicochemical properties of the film (thickness, mechanical properties, moisture, solubility, WVP and antioxidant property), the central composite design (CCD) was used (Table 1 - A). Design Expert-10 software was used to design experiments and analyze data at the 95% probability level and analyze variance and compare averages. In the second part, a factorial design was used at the 95% probability level to investigate the effect of two factors, the amount of methylene blue and vitamin C on antimicrobial properties, surface morphology, etc. (Table 1 -B). To check the capability of the prepared films in identifying oxidizing agents (hydrogen peroxide), films containing methylene blue and vitamin C were used (Table 1 -C). Table 1 A. List of films prepared based on the CCD Film A: Methylene blue (µL) B: Vitamin C (%) 1 50 5.0 2 50 0.0 3 100 0.0 4 50 2.5 5 0 0.0 6 50 2.5 7 100 5.0 8 0 2.5 9 50 2.5 10 0 5.0 11 100 2.5 12 50 2.5 13 50 2.5 Table 1 B. List of films prepared for antimicrobial and antioxidant properties Film A: Methylene blue (µL) B: Vitamin C (%) Control (Pure Cellulose) 0 0 Cel/MB 100 0 Cel/VC 0 5 Cel/MB/VC 100 5 Table 1 C. List of films used as oxidizing agent identification kit Kit A: Methylene blue (µL) B: Vitamin C (%) K1 50 2.5 K2 50 5 K3 100 2.5 K4 100 5 3. Results and Discussion Response surface methodology is a set of statistical techniques and applied mathematics for building empirical models. In response surface designs, the goal is to optimize the output variable (as response). In these designs, the response obtained is affected by several independent variables. In response level designs, building response procedure models is an iterative process. As soon as an approximate model is obtained, it is tested by the goodness of fit method to see if the answer is satisfactory or not. If the answer is not confirmed, the estimation process starts again and more tests are performed. In the design of experiments, the goal is to identify and analyze the variables affecting the outputs with the least number of experiments. In the design of experiments, the goal is to identify and analyze the variables affecting the outputs with the least number of experiments. The methodology is a mathematical-statistical method to optimize the outputs of experiments. In this study, the response surface method was used to investigate the effect of independent variables (methylene blue amount and vitamin C amount) on dependent variables (thickness, mechanical properties, moisture and WVP). Table 2 shows mathematical models obtained from response surface method. In this table, the regression coefficients and adjusted regression coefficients of the obtained mathematical models are also displayed. The probability level of 95% has been used for modeling and checking the effect coefficients of different variables. Table 2 Mathematical models and relationships between independent variables and dependent variables Response Equations R² R² Adj Thickness (mm) =+112.46 + 9.67*A + 7.83*B 0.79 0.75 TS (MPa) =+17.07–3.17*A-2.17*B + 1.00*A*B-0.74*A2 + 2.26*B2 0.89 0.82 EAB (%) =+12.15 + 2.50*A + 1.17*B-2.50*A*B 0.93 0.91 Moisture (%) =+31.38–8.83*A-5.50*B + 3.75*A*B 0.96 0.95 WVP (g µm/m 2 /d/ kPa) =+302.24–79.67*A-20.00*B + 16.75*A*B-34.34*A2 + 8.66*B2 0.97 0.96 3.1. Thickness, TS and EAB The thickness of a biofilm or biomarker affects its mechanical properties. Also, the thickness of a film or membrane affects the permeability to water vapor and other gases. Mechanical resistance in films and markers used for smart food packaging is of great importance. Considering that biomarkers are usually inside the food and in direct contact with the food, it should have a suitable mechanical stability. In the current research, vitamin C and methylene blue used in the structure of composite film have the ability to control the oxidative and microbial spoilage of food products, especially oils, and therefore their thickness and mechanical resistance had to be checked. Figure 2 shows contour plot and perturbation curves of the effect of methylene blue amount and vitamin C percentage on the thickness, TS and EAB of cellulose films. Examining the thickness curves shows that both factors of methylene blue and vitamin C increase the thickness of the film. Considering that cellulosic films have porosity, the placement of methylene blue and vitamin C in these holes as well as the coating of these substances on the surface of cellulosic fibers increases the diameter of cellulosic fibers and ultimately leads to an increase in the thickness of the entire film. According to the results of TS and EAB, both factors, the amount of methylene blue and the percentage of vitamin C, have reduced the tensile strength and have increased the EAB and flexibility of the film. The effect of methylene blue on reducing the tensile strength and increasing the flexibility of the film has been greater than the effect of vitamin C. Due to the presence of N and S groups in methylene blue and O groups in the structure of vitamin C, the probability of electrostatic interactions and hydrogen bonds between these groups with the OH groups of cellulose is high, which makes these interactions of the cohesion of cellulose polymer chains weaker and reduces its tensile strength and probably increases flexibility for this reason. Shi et al. ( 2018 ) modified cellulose film with methylene blue and investigated its structure. The results of their research, from the point of view of the effect of methylene blue on the mechanical properties of cellulose film, confirm the results of the current research to some extent. Also, Atila et al. ( 2022 ) have reported the effect of vitamin C on the physical resistance and other characteristics of cellulosic fibers, and their results show relative agreement with the results of the current research. 3.2. Moisture content and WVP One of the most important reasons for the spoilage of food products is the presence of moisture inside the package. Also, the penetration of unwanted gases and moisture into the food product can affect the shelf life of the food product. The characteristics of water resistance in markers that are used for smart packaging are also very important, because it can affect the sensitivity, efficiency and length of useful use of these markers. Based on this, in this work, the moisture content and water vapor permeability of the prepared composite films were investigated. Figure 3 shows contour plot and perturbation curves of the effect of methylene blue and vitamin C on moisture content and WVP of cellulose films. The results of checking the moisture content show that both methylene blue and vitamin C agents have significantly reduced the moisture content. By examining the chemical structure of cellulose, methylene blue and vitamin C, it can be estimated that N and S groups in methylene blue and O groups in vitamin C have electrostatic interactions with the OH groups of cellulose that this phenomenon decreases the hydrogen interactions between the OH groups of cellulose and the H 2 O molecule, which leads to a decrease in the amount of water molecules on the cellulose surface and the moisture content decreases. Also, both factors of methylene blue and vitamin C have reduced the WVP. As discussed earlier, cellulose has a porous structure that is susceptible to the passage of various gases and especially water vapor molecules, which with the composite of this film with methylene blue and vitamin C, these pores are filled to a large extent and the passage of water molecules is blocked and the WVP is reduced. Also, as discussed in the discussion of thickness, methylene blue and vitamin C increase the thickness of cellulose and thus increase the length of the passage of water molecules, which causes a decrease in permeability to water vapor. Tan et al. ( 2020 ) have investigated the water resistance and antioxidant properties of chitosan-ascorbate film modified with methylene blue. The results of their research in terms of the effect of methylene blue on the characteristics of water vapor permeability and other characteristics of water resistance are in relative agreement with the results of the current research. 3.3. Antioxidant and antibacterial property Methylene blue as a phenothiazine dye has the ability to stain biofilm. This pigment has antimicrobial effects. Methylene blue shows good antibacterial properties by absorbing light. The antibacterial effect of methylene blue is caused by DNA damage, but methylene blue is non-toxic in the human body (Calanna et al., 2019 ). Also, methylene blue has oxazine or antioxidant properties. The antioxidant ability of methylene blue is due to the fact that it loses electrons in the presence of oxidizing agents. Vitamin C is effective in the survival, destruction and overall metabolism of various bacteria. Although bacteria usually have the ability to ferment vitamin C, this vitamin can expose bacteria to oxidative stress and inhibit bacterial growth (Mousavi et al., 2019 ). Considering that methylene blue and vitamin C both have antioxidant and antimicrobial properties, in this study, the antioxidant and antimicrobial properties (against Staphylococcus aureus and Escherichia coli ) of cellulose films containing these two substances have been investigated. Table 3 shows the antioxidant and antibacterial properties of cellulose films containing methylene blue and vitamin C. As it is clear from the results, the pure cellulose film does not have antibacterial properties, but it has a very small amount of antioxidant properties, which is probably related to the physical removal of DPPH radicals by the cellulose film. Both methylene blue and vitamin C have increased antioxidant properties, and the film containing both methylene blue and vitamin C has the highest antioxidant properties. Of course, it is clear that the effect of vitamin C on antioxidant properties is much higher than that of methylene blue. However, vitamin C is a very strong and well-known antioxidant, and the results obtained were expected. Pure cellulose film does not show any antibacterial properties, but films containing vitamin C and methylene blue show good antibacterial properties against both types of bacteria (Gram positive and Gram negative). Cellulose film, which simultaneously contains methylene blue and vitamin C, shows the most antimicrobial properties, which indicates the synergy of the antibacterial effect of methylene blue and vitamin C. There are many studies that have reported the antibacterial properties of methylene blue and vitamin C, which confirm the results of the present study (Thesnaar et al., 2021 ; Mumtaz et al., 2021 ). Table 3 Antioxidant and antibacterial properties of cellulose film and its composites Film Antibacterial activity: Inhibition zone diameter (mm) Antioxidant activity (%) Escherichia coli (G-) Staphylococcus aureus (G+) Cel 0 * 0 4 ± 1 a Cel/MB 6.1 ± 0.4 b 10.2 ± 0.3 b 42 ± 2 c Cel/VC 10.2 ± 0.2 c 12.1 ± 0.4 c 64 ± 3 b Cel/MB/VC 12.8 ± 0.3 c 14.1 ± 0.5 c 82 ± 2 d *Different letters in each column indicate the significance of the difference in means 3.4. SEM images and FTIR spectra Figure 4 shows SEM images and FTIR spectra of cellulose films and its various composites. According to the SEM images, the pure cellulose film has a fibrous structure with dimensions of 20 to 100 nm, which has significant porosity on the surface of the film. By adding methylene blue on the film, the porosity of the film surface has been filled to a large extent and a uniform surface has been created. Vitamin C fills most of the surface of the fibers and in the cellulose film containing vitamin C, the surface porosity of the cellulose film is still observed. In the cellulose film containing methylene blue and vitamin C, the surface of the polymer is largely saturated with the additive and the penetration paths of various gases from the surface of the polymer are largely filled. Acharya et al. ( 2017 ) have investigated the structure of cellulosic film and modified cellulosic films by SEM technique, and their results are in good agreement with the results of the present research (Acharya et al., 2017 ). Examination of FTIR spectra confirms the electrostatic interactions between cellulose fibers, methylene blue and vitamin C. In the FTIR spectrum of pure cellulose film, the functional groups of this polymer have been confirmed by different peaks. In this spectrum, peak at 3330 cm -1 indicates the stretching vibrations of O-H groups. Peak 2894 represents C-H vibrations in R-CH 2 -CH 3 structures. The 1415 peak confirms intra-ring C-C vibrations and the 1152 peak shows C-O stretching vibrations of the C-H bond. Peaks of 1023 and 875 are respectively related to C-O and C-H vibrations connected to different functional groups. By comparing the spectrum of pure cellulose film with its different composites, no significant difference is observed between the spectra because most of the hydrocarbon structure and elements in different composites are similar. But it can be seen that the peaks related to the same functional groups in different composites have appeared in different wave numbers (shifted to higher or lower wave numbers), which indicates the electrostatic interactions between the composite components. Also, in the spectrum of cellulose/methylene blue, a new peak has appeared (compared to the pure cellulose film spectrum) at wave number of 1640, which is related to C-N stretching vibrations, which was expected due to the structure of methylene blue. Also, in the spectrum of cellulose/vitamin C, two new peaks (compared to the pure cellulose film spectrum) have appeared at wave numbers of 1665 and 1751, which are related to C = C stretching vibrations specific to vitamin C. Hishikawa et al. ( 2017 ) investigated the FTIR spectrum of cellulose film, Ovchinnikov et al. ( 2016 ) investigated the chemical structure of methylene blue using FTIR spectrum and Voss et al. ( 2018 ) investigated the FTIR spectrum of polysaccharide film containing vitamin C. The mentioned studies confirm the results of FTIR spectra of the present study. 3.5. XRD spectra and TGA curves Crystalline structure and thermal resistance are characteristics of films that can affect other characteristics of films such as chemical and thermal stability. The type of interaction of additives with cellulose is largely related to the crystalline structure and porosity of cellulose. Figure 5 shows the XRD spectrum and the TGA curve of cellulose films and its various composites. Based on the XRD spectrum, the pure cellulose film has a semi-crystalline cellulose structure, which shows distinct peaks at 2θ of 15, 17 and 22 degrees, and these peaks indicate the crystalline structure of this film, which is in full agreement with the results reported in previous researches. By examining the cellulose film modified with methylene blue and vitamin C, it was found that methylene blue has no significant effect on the crystalline structure of cellulose, but vitamin C increased the crystalline properties of the cellulose film. In the cellulose film containing vitamin C, in addition to the peaks related to cellulose, several peaks at 2θ of 25, 27, 30 and 33 have appeared, all of which are related to the crystal structure of vitamin C. According to the total XRD spectra of different films, it can be concluded that methylene blue does not affect the crystalline nature of cellulose, but vitamin C improves the crystalline order of the cellulose film by coating the cellulose fibers. In the studies of Kumar et al. ( 2018 ) and Ahmed et al. ( 2018 ), the XRD spectra of cellulose and vitamin C have been investigated, which confirm the results reported for the XRD spectra of the peaks recorded for cellulose and vitamin C in this study. Examining the TGA curves showed that the cellulose film undergoes weight decomposition in two stages. The first stage occurs at a temperature between 70 and 130°C, which is related to the evaporation of possible water molecules in the film structure. Weight loss at this stage is about 5%. The second stage of decomposition, which is related to the complete destruction of the polymer, takes place at a temperature between 280 and 400°C. At this stage, 90 to 95% of the polymer is destroyed and destroyed. By examining the TGA curves of composite films, it was found that by adding methylene blue and vitamin C to the film, the temperature of thermal decomposition increases, and in other words, the thermal resistance of the film is improved. The cellulose film containing both methylene blue and vitamin C has the highest decomposition temperature and the highest thermal resistance, which indicates the synergistic effect of these two substances. It can be said that the electrostatic interactions between the composite components, which were also mentioned in the analysis of the FTIR spectra, have led to the improvement of the thermal resistance of the cellulose film. Nurazzi et al. ( 2022 ) have investigated the thermal resistance and crystal structure of cellulose films and its composites, and their results are in good agreement with the results of the present research. 3.6. Absorption rate and release of methylene blue Figure 6 shows the one-factor curve of methylene blue absorption rate on cellulose film and the three-dimensional curve of the effect of the initial amount of methylene blue and vitamin C concentration on the rate of release of methylene blue from cellulose films containing this substance. As it is known, the higher the amount of methylene blue used to prepare the composite film, the higher the amount of absorption on the film, which seems to be a natural result, because the higher the amount of methylene blue, the higher the amount of particles available to the cellulosic fibers. Also, the more methylene blue absorbed on the cellulose film, the higher its release rate will be. An important result that can be seen is that in the composite films where vitamin C is present along with methylene blue in the film, the amount of methylene blue release has decreased a lot. This result is due to the oxidation-reduction interaction of methylene blue and vitamin C, as well as vitamin C, which is added during the production stage after the stabilization of methylene blue on cellulose, acts like a coating and prevents its release. The point that should be noted is that the maximum release of methylene blue from films containing methylene blue and vitamin C was around 15%, which according to some sources reported that methylene blue was not recommended for oral consumption, it seems that this amount of release is problematic, but it should be noted that the methylene blue/vitamin C cellulose film is designed for the intelligent packaging of oily products to delay the oxidation of oils and also show its expiration time, but In this research, the rate of release in water is reported, and it is likely that the rate of release of methylene blue in oils will be much lower. In a similar research, Khakpour et al. ( 2023 ) have used starch film containing lycopene pigment as a nitrite detection kit, and the results of their research in terms of the application and performance of the sensor in identifying oxidants in food products show relative agreement with the results of the current research. 3.7. Application of film as H 2 O 2 sensor (kit) 4 films according to Table 1 -C were used to check the performance of films to identify oxidizing agents (H 2 O 2 ). To use the films as a kit, these films were cut in dimensions of 1×4 cm. In these films, due to the fact that vitamin C is a reducing substance and due to the fact that methylene blue is colorless in its reduced form, the color of these films is white (the main color of cellulose as the base of the kit). Adding oxidizing agents such as hydrogen peroxide to the surface of the kit changes the color of the kit from white to blue. The amount and intensity of color changes of the sensor depend on the concentration of the oxidizing agent. Therefore, a mathematical relationship is established between the concentration of the oxidizing agent and the color changes, through which the prepared kits can be calibrated with respect to the oxidizing agents, and the prepared calibration curve can be used to obtain the amount and concentration of the oxidizing agent. In this study, hydrogen peroxide was used as a soft oxidizer to investigate the behavior of the prepared sensors. Figure 1 -B shows the color changes of the sensor in different concentrations of hydrogen peroxide. Table 4 shows the figure of merits of the 4 prepared kits compared to hydrogen peroxide. As it is clear from the results of this table, the cellulose film containing the highest amount of methylene blue and the highest amount of vitamin C shows the highest sensitivity to hydrogen peroxide oxidant. It is known that by increasing the amount of methylene blue on the surface of the film, the amount of this active substance that is available to the oxidant also increases, and causes even small amounts of the oxidant to cause significant color changes on the surface of the film, which causes The detection limit of the sensor to measure the oxidant should also be reduced. It should be mentioned that in the examination of sensors, the lower the detection limit of the sensor is, it indicates the high detection power of the sensor and the high sensitivity of the sensor. Jiang et al. ( 2013 ) have used silica/cellulose composite modified with catalytic enzymes as a sensor to measure hydrogen peroxide. Considering the use of enzyme and catalytic structure in the mentioned study, it should be noted that the present research has presented a simpler, cheaper and more accessible system compared to the research of Jiang et al. Also, Jiang et al.'s results show a good match with the results of the current research in terms of the application of the sensor and the functional system of the sensor. Table 4 Figure of merits of the H 2 O 2 Kits based on the b (color factor) compared to the H 2 O 2 concentration Kit DL (mg/100ml) LR (mg/100ml) S (100ml/mg) R 2 K1 (Cel/MB50/VC2.5) 6.12 6.12-22 0.1549 0.997 K2 (Cel/MB50/VC5) 8.4 8.4–25 0.1742 0.998 K3 (Cel/MB100/VC2.5) 2 2–21 0.2526 0.998 K4 (Cel/MB100/VC5) 1.9 2.1–20 0.2995 0.999 DL: Detection limit, LR: Liner range, S: Sensitivity, R 2 : Coefficient of determination 4. Conclusion In this research, an active and smart biodegradable film was designed. For this purpose, cellulose nanofiber film modified with methylene blue and vitamin C. In this research, hydrogen peroxide was used as a soft oxidant to investigate the sensing behavior of the prepared cellulose film. Using mathematical equations, linear relationships were established between the color changes of the sensor and the concentration of hydrogen peroxide, and it was used for qualitative and quantitative measurement of hydrogen peroxide. Examination of the thickness showed that both the amount of methylene blue and vitamin C increased the thickness of the film. Methylene blue and vitamin C decreased the tensile strength and increased EAB. Methylene blue and vitamin C agents significantly reduced the moisture content. Also, both factors reduced the amount of methylene blue and vitamin C decreased WVP. The pure cellulose film did not have antibacterial properties, but it had antioxidant properties to a very small extent. Both methylene blue and vitamin C increased the antioxidant properties. The pure cellulose film did not show any antimicrobial properties, but the films containing vitamin C and methylene blue showed good antimicrobial properties against both types of bacteria (Gram positive and Gram negative). The pure cellulose film had a fibrous structure with dimensions of 20 to 100 nm. By adding methylene blue on the film, the porosity of the film surface was filled to a large extent and a uniform surface was created. Examination of FTIR spectra confirmed the electrostatic interactions between cellulose fibers, methylene blue and vitamin C. Methylene blue had no significant effect on the crystalline structure of cellulose, but vitamin C increased the crystalline properties of cellulose film. The electrostatic interactions between the composite components, which were also proven in the FTIR spectrum analysis, have led to the improvement of the thermal resistance of the cellulose film. The highest rate of release of methylene blue from films containing methylene blue and vitamin C was about 15%. Cellulose film containing the highest amount of methylene blue and the highest amount of vitamin C showed the highest sensitivity to hydrogen peroxide oxidant. The prepared sensors were able to measure hydrogen peroxide with appropriate sensitivity and acceptable detection limits. Due to this feature, the prepared films will be used to detect the oxidation process in smart food packaging. Declarations Ethical Approval Ethical approval for the study was obtained from the relevant local ethics committees. Consent to Participate All authors consent to participate in the research project Consent to Publish All authors consent to publish the current manuscript. Conflict of interest There is not any Conflict of interest between authors. Funding Declaration Author Contribution Sajad Pirsa conceived of the presented idea, Narmela Asefi developed the theory and performed the computations. Sajad Pirsa verified the analytical methods. Sina Sadeghi discussed the results and contributed to the final manuscript. Sina Sadeghi out the experiment. Sajad Pirsa and Mehdi Gharekhani wrote the manuscript and revised it. Data Availability The data that support the findings of this study are available from the corresponding author upon reasonable request. References Abdolsattari, P., Peighambardoust, S. J., Pirsa, S., Fasihnia, S. H. & Peighambardoust, S. H. Investigating microbial properties of traditional Iranian white cheese packed in active LDPE films incorporating metallic and organoclay nanoparticles. Chem. Rev. Lett. 3 (4), 168–174 (2020). Abdolsattari, P., Rezazadeh-Bari, M. & Pirsa, S. Smart film based on polylactic acid, modified with polyaniline/ZnO/CuO: Investigation of physicochemical properties and its use of intelligent packaging of orange juice. Food Bioprocess Technol. 15 (12), 2803–2825 (2022). Acharya, S., Hu, Y., Moussa, H. & Abidi, N. Preparation and characterization of transparent cellulose films using an improved cellulose dissolution process. J. Appl. Polym. Sci. , 134 (21). (2017). Ahmed, I. et al. Vitamin C/stearic acid hybrid monolayer adsorption at air–water and air–solid interfaces. ACS omega . 3 (11), 15789–15798 (2018). Alizadeh, S., Pirsa, S. & Amiri, S. Development of a colorimetric sensor based on nanofiber cellulose film modified with ninhydrin to measure the formalin index of fruit juice. International Journal of Biological Macromolecules, 253, p.127035. (2023). Atila, D., Karataş, A., Keskin, D. & Tezcaner, A. Pullulan hydrogel-immobilized bacterial cellulose membranes with dual-release of vitamin C and E for wound dressing applications. Int. J. Biol. Macromol. 218 , 760–774 (2022). Calanna, F. et al. Debridement, antibiotic pearls, and retention of the implant (DAPRI): a modified technique for implant retention in total knee arthroplasty PJI treatment. Journal of Orthopaedic Surgery, 27(3), p.2309499019874413. (2019). Chiarappa, G. et al. Mathematical modeling of L-(+)-ascorbic acid delivery from pectin films (packaging) to agar hydrogels (food). J. Food Eng. 234 , 73–81 (2018). Colnik, M., Knez-Hrnčič, M., Škerget, M. & Knez, Ž. Biodegradable polymers, current trends of research and their applications, a review. Chem. Ind. Chem. Eng. Q. 26 (4), 401–418 (2020). Erfani, A., Pirouzifard, M. K. & Pirsa, S. Photochromic biodegradable film based on polyvinyl alcohol modified with silver chloride nanoparticles and spirulina; investigation of physicochemical, antimicrobial and optical properties. Food Chemistry, 411, p.135459. (2023). Evora, P. R. B. et al. Twenty years of vasoplegic syndrome treatment in heart surgery. Methylene blue revised. Revista Brasileira de Cirurgia Cardiovasc. 30 (1), 84–92 (2015). Farooq, A. et al. Cellulose from sources to nanocellulose and an overview of synthesis and properties of nanocellulose/zinc oxide nanocomposite materials. Int. J. Biol. Macromol. 154 , 1050–1073 (2020). Gupta, P. K. et al. An update on overview of cellulose, its structure and applications. Cellulose, 201(9), p.84727. (2019). Hishikawa, Y., Togawa, E. & Kondo, T. Characterization of individual hydrogen bonds in crystalline regenerated cellulose using resolved polarized FTIR spectra. ACS omega . 2 (4), 1469–1476 (2017). Jiang, Y. et al. Enzyme-mimetic catalyst-modified nanoporous SiO2–cellulose hybrid composites with high specific surface area for rapid H2O2 detection. ACS Appl. Mater. Interfaces . 5 (6), 1913–1916 (2013). Khakpour, F., Pirsa, S. & Amiri, S. Modified Starch/CrO/Lycopene/Gum Arabic Nanocomposite Film: Preparation, Investigation of Physicochemical Properties and Ability to Use as Nitrite Kit. J. Polym. Environ. 31 (9), 3875–3893 (2023). Khwaldia, K. Physical and mechanical properties of hydroxypropyl methylcellulose–coated paper as affected by coating weight and coating composition. BioResources . 8 (3), 3438–3452 (2013). Kumar, T. S. M. et al. All-cellulose composite films with cellulose matrix and Napier grass cellulose fibril fillers. Int. J. Biol. Macromol. 112 , 1310–1315 (2018). Lin, X., Ni, Y. & Kokot, S. An electrochemical DNA-sensor developed with the use of methylene blue as a redox indicator for the detection of DNA damage induced by endocrine-disrupting compounds. Anal. Chim. Acta . 867 , 29–37 (2015). Liu, Y. et al. A review of cellulose and its derivatives in biopolymer-based for food packaging application 112pp.532–546 (Trends in Food Science & Technology, 2021). Mousavi, S., Bereswill, S. & Heimesaat, M. M. Immunomodulatory and antimicrobial effects of vitamin C. Eur. J. Microbiol. Immunol. 9 (3), 73–79 (2019). Mumtaz, S. et al. Evaluation of antibacterial activity of vitamin C against human bacterial pathogens. Brazilian J. Biology . 83 , e247165 (2021). Mustafa, F. & Andreescu, S. Chemical and biological sensors for food-quality monitoring and smart packaging. Foods, 7(10), p.168. (2018). Nurazzi, N. M. et al. Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) of PLA/cellulose composites. In Polylactic acid-based nanocellulose and cellulose composites (145–164). CRC. (2022). Ovchinnikov, O. V. et al. Manifestation of intermolecular interactions in FTIR spectra of methylene blue molecules 86pp.181–189 (Vibrational Spectroscopy, 2016). Pehlivan, F. E. Vitamin C: An antioxidant agent. Vitam. C . 2 , 23–35 (2017). Perni, S. et al. Antibacterial activity of light-activated silicone containing methylene blue and gold nanoparticles of different sizes. J. Cluster Sci. 21 , 427–438 (2010). Pirsa, S. & Mohammadi, B. Conducting/biodegradable chitosan-polyaniline film; Antioxidant, color, solubility and water vapor permeability properties. Main Group Chem. 20 (2), 133–147 (2021). Pirsa, S., Mahmudi, M. & Ehsani, A. Biodegradable film based on cress seed mucilage, modified with lutein, maltodextrin and alumina nanoparticles: Physicochemical properties and lutein controlled release. Int. J. Biol. Macromol. 224 , 1588–1599 (2023). Reis, D. T., dos Santos Pereira, A. K., Scheidt, G. N. & Pereira, D. H. Plant and bacterial cellulose: production, chemical structure, derivatives and applications pp.321–329 (The Electronic Journal of Chemistry, 2019). Seroglazova, A. S. et al. Ox/Red-controllable combustion synthesis of foam-like PrFeO3 nanopowders for effective photo-Fenton degradation of methyl violet. Advanced Powder Technology, 33(2), p.103398. (2022). Shabkhiz, M. A., Pirouzifard, M. K., Pirsa, S. & Mahdavinia, G. R. Alginate hydrogel beads containing Thymus daenensis essential oils/Glycyrrhizic acid loaded in β-cyclodextrin. Investigation of structural, antioxidant/antimicrobial properties and release assessment. Journal of Molecular Liquids, 344, p.117738. (2021). Shi, C., Tao, F. & Cui, Y. Evaluation of nitriloacetic acid modified cellulose film on adsorption of methylene blue. Int. J. Biol. Macromol. 114 , 400–407 (2018). Tan, W. et al. Preparation and physicochemical properties of antioxidant chitosan ascorbate/methylcellulose composite films. Int. J. Biol. Macromol. 146 , 53–61 (2020). Thesnaar, L., Bezuidenhout, J. J., Petzer, A., Petzer, J. P. & Cloete, T. T. Methylene blue analogues: In vitro antimicrobial minimum inhibitory concentrations and in silico pharmacophore modelling. European Journal of Pharmaceutical Sciences, 157, p.105603. (2021). Voss, G. T. et al. Polysaccharide-based film loaded with vitamin C and propolis: A promising device to accelerate diabetic wound healing. Int. J. Pharm. 552 (1–2), 340–351 (2018). Yorghanlu, R. A., Hemmati, H., Pirsa, S. & Makhani, A. Production of biodegradable sodium caseinate film containing titanium oxide nanoparticles and grape seed essence and investigation of physicochemical properties. Polym. Bull. 79 (10), 8217–8240 (2022). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 02 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 02 Jan, 2025 Reviews received at journal 18 Nov, 2024 Reviews received at journal 17 Nov, 2024 Reviews received at journal 15 Nov, 2024 Reviewers agreed at journal 07 Nov, 2024 Reviewers agreed at journal 05 Nov, 2024 Reviewers agreed at journal 05 Nov, 2024 Reviewers agreed at journal 05 Nov, 2024 Reviewers invited by journal 05 Nov, 2024 Editor assigned by journal 05 Nov, 2024 Editor invited by journal 04 Nov, 2024 Submission checks completed at journal 30 Oct, 2024 First submitted to journal 16 Oct, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5274857","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":375333632,"identity":"da15aebe-8d23-4cf7-9414-136e56137ca3","order_by":0,"name":"Sina Sadeghi","email":"","orcid":"","institution":"Islamic Azad University","correspondingAuthor":false,"prefix":"","firstName":"Sina","middleName":"","lastName":"Sadeghi","suffix":""},{"id":375333633,"identity":"2105732a-bc81-4499-97ed-8f5010c9ac87","order_by":1,"name":"Sajad 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University","correspondingAuthor":false,"prefix":"","firstName":"Mehdi","middleName":"","lastName":"Gharekhani","suffix":""}],"badges":[],"createdAt":"2024-10-16 10:08:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5274857/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5274857/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-07566-6","type":"published","date":"2025-07-02T15:58:45+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":69439620,"identity":"65ef6b09-7e36-4c5b-a94b-445d34110ee1","added_by":"auto","created_at":"2024-11-20 11:07:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2102328,"visible":true,"origin":"","legend":"\u003cp\u003eCellulose films modified with methylene blue and vitamin C (A), the effect of hydrogen peroxide on the color changes of Cel/MB/VC film (B) and the system for measuring color characteristics (C)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5274857/v1/847a5cae78d88c2d34e2f903.png"},{"id":69439623,"identity":"58ec2221-cb86-4fac-a3dc-246b7692f795","added_by":"auto","created_at":"2024-11-20 11:07:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":201026,"visible":true,"origin":"","legend":"\u003cp\u003eContour plot and perturbation curves of the effect of methylene blue and vitamin C on thickness, TS and EAB of cellulose films\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5274857/v1/9afbb7aff7470d064a52c238.png"},{"id":69439622,"identity":"6cd020a1-a657-4aa2-bed1-a69ffecfbbc2","added_by":"auto","created_at":"2024-11-20 11:07:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":130525,"visible":true,"origin":"","legend":"\u003cp\u003eContour plot and perturbation curves of the effect of methylene blue and vitamin C on moisture content and WVP of cellulose films\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5274857/v1/0f7300f9b84a8f2d5ba9c457.png"},{"id":69439621,"identity":"7f801ccf-e464-4cc0-8b1a-a95bcc963b98","added_by":"auto","created_at":"2024-11-20 11:07:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1221263,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images (A) and FTIR spectra (B) of cellulose films and its various composites\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5274857/v1/09da38dae4923a46dc56baa1.png"},{"id":69439625,"identity":"835e6448-b4e4-402d-9b62-822d4d59b7e6","added_by":"auto","created_at":"2024-11-20 11:07:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":294952,"visible":true,"origin":"","legend":"\u003cp\u003eXRD spectra (A) and TGA curves (B) of cellulose films and its various composites\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5274857/v1/01c509faa3cc67c9ecbf6c07.png"},{"id":69440802,"identity":"e325a230-8316-4477-ac10-abd45169dc29","added_by":"auto","created_at":"2024-11-20 11:15:56","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":53477,"visible":true,"origin":"","legend":"\u003cp\u003eOne-factor curve of the effect of methylene blue on the methylene blue absorption rate and three-dimensional curve of the effect of the initial amount of methylene blue and vitamin C on the rate of methylene blue release\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5274857/v1/02e9b5af5df54c7d13c4f51b.png"},{"id":86180553,"identity":"160c8f77-de2e-4126-9e9f-c98ddcdfbab2","added_by":"auto","created_at":"2025-07-07 16:22:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5008121,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5274857/v1/a9f1fb57-6714-4022-9c59-170b1dc67172.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Biodegradable film based on cellulose nanofiber/methylene blue/vitamin C: investigation of physicochemical properties and usability as a kit for identification of oxidants (hydrogen peroxide)","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe release of synthetic and petroleum polymers into the environment causes environmental pollution and creates many biological problems for humans and animals. Due to the fact that these polymers are not easily decomposed, the use of these polymers in various industries, including food packaging, faces many problems. Therefore, in recent years, the use of biodegradable polymers in food packaging has become very popular. Biodegradable polymers of plant and biological origin can be a good substitute for petroleum polymers. While these polymers have suitable physical and chemical properties for food packaging, they are easily decomposed when released in the environment and do not have environmental pollution problems (Pirsa and Mohammadi, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Colnik et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Pirsa et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Biopolymers with protein, polysaccharide and lipid structures or their composites are used to produce biodegradable plastics. Cellulose and its derivatives are of special importance among different biological sources for the preparation of biodegradable films. Because cellulose is found in abundance in nature and is relatively cheap and available, and its structure can be modified easily (Liu et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Yorghanlu et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Farooq et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Erfani et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCellulose is an organic compound that is known as the most abundant biopolymer on earth. Cellulose is a complex carbohydrate or polysaccharide consisting of hundreds to thousands of glucose molecules that are linked together and form a chain. Unlike animals, plants, algae and some bacteria and other organisms have the ability to produce this substance. Cellulose is the main structural molecule in the cell walls of plants and algae. Cellulose has many important derivatives. Most of them are biodegradable and renewable resources. Cellulose-derived compounds are usually considered non-toxic and non-allergenic. Derivatives include cellulose nanofiber, celluloid, cellulose acetate, methylcellulose, hydroxypropylmethylcellulose and carboxymethylcellulose, all of which are used in the preparation of biodegradable films (Gupta et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Reis et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMethylene blue is a functional dye that acts as a red-ox indicator and has different colors (blue/colorless) in oxidized or reduced states. One of the tests to determine the quality of milk, with which the number of bacteria in raw milk is estimated, is the methylene blue regeneration test. Chemically, methylene blue is a crystalline substance and a type of organic chloride salt, which is included among thiazine dyes. Thiazine dye means that it has oxazine or antioxidant properties (Seroglazova et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Methylene blue is often used in textile industries, aquariums, medical, dental and chemical laboratories. Another property of methylene blue is the ability to protect the heart and vascular system (Evora et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). One of the most important uses of methylene blue is its use as a disinfectant in aquariums. Aquariums are often infected with fungi and infectious bacteria, which endangers not only the health of the fish, but also the eggs of the fish. In these cases, by adding methylene blue to the aquarium water, fungal infections and bacteria are eliminated (Lin et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Perni et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eVitamin C is an antioxidant (or regenerative agent) that has the ability to prevent damage from free radicals and dangerous chemicals such as cigarette smoke. Free radicals play a role in the occurrence of some diseases, including cancer, heart disease and arthritis. This vitamin protects the skin from the harmful effects of the ultraviolet rays of the sun. Also, this vitamin helps to increase the immunity of the body and the strength of gums and teeth. This vitamin also makes collagen (the strongest part of connective tissue that holds all body parts together) and is effective in preventing blood cholesterol from rising and blood clots in the veins (Pehlivan, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Chiarappa et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eActive and smart packaging are among the new packaging methods that have attracted the attention of many scientists (Abdolsattari et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Shabkhiz et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Kits (color sensors) sensitive to the concentration of carbon dioxide, labels sensitive to the amount of oxygen, time-temperature labels and labels sensitive to changes in the pH of food are among the kits that are recently used in the smart packaging of food products (Abdolsattari et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Packages containing smart kits, due to having special identifiers, can detect environmental conditions and changes in food and provide consumers with information about the quality and whether the food is healthy or unhealthy by means of these indicators. Smart food packaging does not work directly to increase the shelf life of food, but rather provides information about food quality to stakeholders in food supply chains (Mustafa and Andreescu, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this research, an active and intelligent biodegradable film was designed that, in addition to being able to delay the oxidation of food and increase its shelf life, at the same time, it can intelligently monitor the oxidation process in food products and increase its shelf life and estimate its expiration date. For this purpose, cellulose nanofiber film modified with methylene blue and vitamin C was used to prepare smart film. Methylene blue has a blue color in its natural state, which becomes colorless in the composite with vitamin C (as a reducing agent). Therefore, cellulose film modified with methylene blue and vitamin C is a white film. This film has the ability to detect the oxidizing agent. In this research, hydrogen peroxide was used as a soft oxidant to investigate the sensing behavior of the prepared cellulose film. Using mathematical equations, linear relationships were established between the color changes of the sensor and the concentration of hydrogen peroxide, and it was used for qualitative and quantitative measurement of hydrogen peroxide. The obtained results showed that the cellulose film modified with methylene blue and vitamin C has suitable physicochemical properties to be used as a smart marker in food packaging and can identify and measure hydrogen peroxide as a smart marker.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Chemicals\u003c/h2\u003e \u003cp\u003eCellulose film with Nano fibrous structure (thickness 30 to 100 nm) and porosity 5 to 10 micrometers (molecular weight 100,000 to 200,000 Daltons) was obtained from Zardab Tabriz (Iran). Hydrogen peroxide 35%, methylene blue, vitamin C, sodium hydroxide, silica gel and other chemical compounds used were obtained from Merck (Germany) and Aldrich (USA) companies and were used without further purification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Preparation of cellulose/methylene blue/vitamin C film\u003c/h2\u003e \u003cp\u003eTo prepare standard methylene blue solution, 500 mg of commercial methylene blue powder was dissolved in 1 liter of distilled water. Dissolution was continued at room temperature while stirring for 10 minutes. In order to prepare films, first, cellulose films were prepared in dimensions of 10\u0026times;10 cm. In a 100 ml beaker, 50 ml of distilled water was added and some methylene blue solution was added to it at three levels (0, 50 and 100 \u0026micro;L - according to the Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e1\u003c/span\u003e-A) and dissolved. Next, the cellulose film was immersed in the prepared solution for 10 minutes. After 10 minutes, the blue film was removed from the solution and dried in an oven at 50\u0026deg;C. Then the colored film was immersed in 50 ml of 5% NaOH solution for 10 minutes. Then the film was removed from the solution and dried again in the oven at 50\u0026deg;C. The dried film was immersed in a solution containing vitamin C at three levels (0, 2.5 and 5% - according to the Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e1\u003c/span\u003e-A) for 10 minutes. At this stage, the color of the film turned white. At the end, the film was removed from the solution and dried in an oven at 50\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e-a). The dried films were stored in black plastic bags at refrigerator temperature until the tests were performed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Physical and chemical characteristics of the prepared films\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1. Thickness\u003c/h2\u003e \u003cp\u003eThe thickness of each film was measured at 5 different points. The obtained numbers were averaged and reported. The thickness of the film was used to determine mechanical resistance and water resistance, etc. In this test, a digital micrometer (Insize Digital Outside Micrometer 3108-25A) was used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2. Mechanical characteristics\u003c/h2\u003e \u003cp\u003eThe mechanical properties of the films include elongation at breaking point (EAB) and tensile strength (TS) are important factors for food packaging. To check the mechanical properties, the samples were conditioned for 24 hours in special conditions. Special conditions included relative humidity RH\u0026thinsp;=\u0026thinsp;55%. The films were cut with a special cutter in dimensions of 1\u0026times;8 cm in a dumbbell shape. The films were placed between the two jaws of the device, and the initial distance between the two jaws is 50 mm. The upper jaw was moved relative to the lower jaw at a speed of 5 mm/min. The mechanical properties were recorded by a computer. A Texture analyzer-brand histometer (Zwick/Roell model FR010, Germany) was used to perform this test.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.3.3. Moisture content\u003c/h2\u003e \u003cp\u003eTo measure the moisture of the films, first the films was cut into 3\u0026times;3 cm dimensions. Then, the cut films were kept in a desiccator for 24 hours in specific humidity conditions (55% relative humidity) at a temperature of 25\u0026deg;C. The desiccator contained silica gel. At this stage, the weight of the films was measured and recorded as the initial weight. Then, in order to completely remove the moisture from the films, the film sample was heated at 100\u0026deg;C for 2 hours and finally its final weight was measured and recorded. The following equation was used to calculate the moisture content of the film.\u003c/p\u003e \u003cp\u003eHumidity (%) =\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\frac{{\\varvec{W}}_{\\varvec{i}}-{\\varvec{W}}_{\\varvec{f}}}{{\\varvec{W}}_{\\varvec{i}}}\\)\u003c/span\u003e\u003c/span\u003e \u0026times;100 (1)\u003c/p\u003e \u003cp\u003eWi: Initial film weight\u003c/p\u003e \u003cp\u003eWf: Final film weight\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.3.4. Water vapor permeability (WVP)\u003c/h2\u003e \u003cp\u003ePermeability relative to water vapor was measured by gravimetric method. For this test, the test film was sealed in a glass vial with a height of 4 cm containing silica gel to maintain zero percent relative humidity (RH 0%) in the vial. The vials had an inner diameter of 6.4 cm and their outer diameter was 8.9 cm. Also, the exposed surface of the vials was 26 cm\u003csup\u003e2\u003c/sup\u003e. The vials were placed in a controlled temperature (38\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C) and relative humidity (90\u0026thinsp;\u0026plusmn;\u0026thinsp;3%). The transfer of water vapor was determined from the increase in the weight of the vial. Changes in cell weight were recorded as a function of time. The slope of weight changes versus time (after reaching steady state) was calculated by linear regression. Water vapor transmission rate (WVTR) was defined as the slope (g/d) divided by the transfer area (m\u003csup\u003e2\u003c/sup\u003e). After penetration tests, WVP was calculated as follows (Khwaldia, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2013\u003c/span\u003e):\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:WVP=\\frac{WVTR\\times\\:X}{\\varDelta\\:p}\\)\u003c/span\u003e \u003c/span\u003e [g \u0026micro;m/m\u003csup\u003e2\u003c/sup\u003e /d/ kPa] (2)\u003c/p\u003e \u003cp\u003eWhere X is the thickness of the paper and Δp is the partial pressure difference of water vapor throughout the film and its numerical value is 9.5 kPa.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.3.5. Antioxidant property\u003c/h2\u003e \u003cp\u003eDPPH (2,2-diphenyl-1-picrylhydrazy) radical quenching method was used to measure the antioxidant power of the films. For this test, to prepare film extract, 25 mg of each film was dissolved in 4 ml of distilled water for 2 minutes. Then, 2.8 ml of film extract solution was mixed with 1 mM methanolic DPPH solution (0.2 ml). The obtained solution was stirred with a vortex 2000 rpm for 2 minutes and kept in a dark place for 1 hour. The absorbance of the solution was recorded using a spectrophotometer (Model T60 UV, USA) at a wavelength of 517 nm. The following equation was used to calculate the antioxidant percentage.\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\text{Antioxidant activity (%)}=\\frac{{A}_{b}-{A}_{s}}{{A}_{b}}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e3\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eA\u003csub\u003eb\u003c/sub\u003e: Absorption rate of the control sample (DPPH methanolic solutions: 1 mM)\u003c/p\u003e \u003cp\u003eA\u003csub\u003es\u003c/sub\u003e: Sample absorption rate\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.3.6. Antibacterial activity\u003c/h2\u003e \u003cp\u003eAgar diffusion method was used to determine the antibacterial property of the films. For this purpose, the films were cut into discs (with a diameter of 15 mm) and then placed on Mueller Hinton agar plates containing \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC6538 and \u003cem\u003eEscherichia Coli\u003c/em\u003e ATCC13706, with a concentration of (10\u003csup\u003e7\u003c/sup\u003e CFU/mL). took The plates were incubated for 24 hours at 37\u0026deg;C. After 24 hours, the radius of bacterial non-growth halos around the films (in millimeters) was measured with a precision caliper.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.3.7. Scanning electron microscopy (SEM)\u003c/h2\u003e \u003cp\u003eThe technique of scanning electron microscope (ZEISS, SIGMA, Germany) was used to investigate the polymer structure, surface characteristics, porosity and particle distribution of the prepared films. In this method, the surface of the film samples was covered with a thin layer of gold before analysis. The accelerator voltage of 20 kV was used for the adjustment device. Imaging of the surface of the samples was done with different magnifications after placing the films in the special position of the sample in the device.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.3.8. Fourier Transform Infrared Spectroscopy (FTIR)\u003c/h2\u003e \u003cp\u003eFTIR device (Tensor 27, Bruker, Germany) was used to investigate physical or chemical interactions between film components. To perform this test, first the films was dried and completely powdered. The obtained powder was mixed with KBr at a ratio of 1 to 20. The powder mixture was turned into a thin film with a special pressing machine. FTIR spectra of each sample were recorded separately. The spectrum of the samples was recorded with a resolution of 4 cm\u003csup\u003e-1\u003c/sup\u003e in the range of 400\u0026ndash;4000 cm\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.3.9. X-ray diffraction (XRD)\u003c/h2\u003e \u003cp\u003eXRD technique was used with the help of X-ray diffractometer (Kristalloflex D500, Siemens, Germany) to check the crystalline or amorphous structure. In this technique, first, the film samples were placed in the special position of the device (sample cell). The primary rays were irradiated to the sample at ambient temperature and the reflected rays were collected in the range of angle 2θ\u0026thinsp;=\u0026thinsp;0\u0026ndash;80\u0026deg;. The XRD spectra of the samples were drawn automatically by the machine. Cu Kα radiation source was used at a wavelength of 0.154 nm. The specifications of the device include X-ray generator at 40 kV and 40 mA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.3.10. Thermogravimetric analysis (TGA)\u003c/h2\u003e \u003cp\u003eTo check the thermal stability of the films, the TGA test was used by a thermal analyzer (Linseis \u0026ndash; L81A1750, Germany). For this purpose, films were prepared in the form of 10 mg samples. The film samples were heated under a nitrogen atmosphere of 50 cm\u003csup\u003e3\u003c/sup\u003e/min in aluminum cups in the temperature range of 30\u0026ndash;600\u0026deg;C. The heating rate was 10\u0026deg;C/min. The empty aluminum cup was considered as a reference and the TGA curve was drawn and recorded by the device.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Absorption and release of methylene blue\u003c/h2\u003e \u003cp\u003eConsidering that the UV-Vis spectrum of methylene blue has maximum absorption at three wavelengths of 250, 300 and 665 nm, and the maximum absorption is at the wavelength of 665 nm, the wavelength of 665 nm was used to investigate the absorption and release of methylene blue. To check the absorption of methylene blue, the UV-Vis spectrum of the solutions used to prepare the film was measured before and after the absorption of methylene blue by the film, and the amount of absorption was calculated from the decrease in the intensity of the absorption peak at the wavelength of 665 nm (Eq.\u0026nbsp;4). To check the release rate, the films containing methylene blue were immersed in 100 ml of distilled water and after 24 hours, the absorbance of the solution was measured at the wavelength of 665, and the release rate of methylene blue was checked from Eq.\u0026nbsp;5.\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:Absorption\\:\\left(\\%\\right)=\\frac{{A}_{\\text{M}\\text{B}1}-{A}_{\\text{M}\\text{B}2}}{{A}_{\\text{M}\\text{B}1}}\\)\u003c/span\u003e \u003c/span\u003e\u0026times;100 (4)\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:Release\\:\\left(\\%\\right)=\\frac{{A}_{\\text{M}\\text{B}\\text{i}}-{A}_{\\text{M}\\text{B}\\text{r}}}{{A}_{\\text{M}\\text{B}\\text{i}}}\\)\u003c/span\u003e \u003c/span\u003e\u0026times;100 (5)\u003c/p\u003e \u003cp\u003eIn this regard, A\u003csub\u003eMB1\u003c/sub\u003e is the absorption rate of methylene blue solution before coating on cellulose film and A\u003csub\u003eMB2\u003c/sub\u003e is the absorption rate of methylene blue solution after coating on cellulose film. Also, A\u003csub\u003eMBi\u003c/sub\u003e is the absorption of the initial solution (standard) of methylene blue, and A\u003csub\u003eMBr\u003c/sub\u003e is the absorption of the released solution of methylene blue.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Cel/MB/VC film sensing behavior\u003c/h2\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1. Color characteristics of the sensor\u003c/h2\u003e \u003cp\u003eIn this step, the color factors of the used films were analyzed. Considering that the film changed from white to blue, factor b was further investigated. In this factor, negative numbers indicate the intensity of the blue color, and as the number moves towards more positive numbers, the blue color decreases. To record color features, the system designed in previous studies was used (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e-c) (Alizadeh et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e2.5.2. Calibrating the sensor and sensing H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/h2\u003e \u003cp\u003eHydrogen peroxide was used as a soft oxidant to calibrate the sensor and check the response of the sensor to oxidizing agents. Based on this, different concentrations of hydrogen peroxide were prepared and placed in contact with the sensor, and the response of the sensor was calculated with the Eq.\u0026nbsp;\u003cspan refid=\"Equ2\" class=\"InternalRef\"\u003e6\u003c/span\u003e. Finally, the calibration curve of the sensor was drawn as the response of the sensor to different concentrations of hydrogen peroxide and the figures of merit of the sensor were calculated.\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$\\:Response=\\frac{{b}_{1}-{b}_{0}}{{b}_{0}}\\times\\:100$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e6\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eIn this regard, b\u003csub\u003e0\u003c/sub\u003e is the value of b factor before contact with hydrogen peroxide and b\u003csub\u003e1\u003c/sub\u003e is the value of b factor after contact with hydrogen peroxide.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Statistical analysis\u003c/h2\u003e \u003cp\u003eThe statistical analysis and analysis of the data obtained in this research was done in three parts. In order to examine the effect of two factors, the amount of methylene blue and vitamin C on the physicochemical properties of the film (thickness, mechanical properties, moisture, solubility, WVP and antioxidant property), the central composite design (CCD) was used (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e1\u003c/span\u003e- A). Design Expert-10 software was used to design experiments and analyze data at the 95% probability level and analyze variance and compare averages. In the second part, a factorial design was used at the 95% probability level to investigate the effect of two factors, the amount of methylene blue and vitamin C on antimicrobial properties, surface morphology, etc. (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e1\u003c/span\u003e-B). To check the capability of the prepared films in identifying oxidizing agents (hydrogen peroxide), films containing methylene blue and vitamin C were used (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e1\u003c/span\u003e-C).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eA. List of films prepared based on the CCD\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFilm\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA: Methylene blue (\u0026micro;L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eB: Vitamin C (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e11\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e12\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e13\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eB. List of films prepared for antimicrobial and antioxidant properties\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFilm\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA: Methylene blue (\u0026micro;L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eB: Vitamin C (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eControl (Pure Cellulose)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCel/MB\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCel/VC\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCel/MB/VC\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eC. List of films used as oxidizing agent identification kit\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKit\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA: Methylene blue (\u0026micro;L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eB: Vitamin C (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eK1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eK2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eK3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eK4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cp\u003eResponse surface methodology is a set of statistical techniques and applied mathematics for building empirical models. In response surface designs, the goal is to optimize the output variable (as response). In these designs, the response obtained is affected by several independent variables. In response level designs, building response procedure models is an iterative process. As soon as an approximate model is obtained, it is tested by the goodness of fit method to see if the answer is satisfactory or not. If the answer is not confirmed, the estimation process starts again and more tests are performed. In the design of experiments, the goal is to identify and analyze the variables affecting the outputs with the least number of experiments. In the design of experiments, the goal is to identify and analyze the variables affecting the outputs with the least number of experiments. The methodology is a mathematical-statistical method to optimize the outputs of experiments. In this study, the response surface method was used to investigate the effect of independent variables (methylene blue amount and vitamin C amount) on dependent variables (thickness, mechanical properties, moisture and WVP). Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows mathematical models obtained from response surface method. In this table, the regression coefficients and adjusted regression coefficients of the obtained mathematical models are also displayed. The probability level of 95% has been used for modeling and checking the effect coefficients of different variables.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMathematical models and relationships between independent variables and dependent variables\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eResponse\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEquations\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR\u0026sup2;\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eR\u0026sup2;\u003csub\u003eAdj\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThickness (mm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e=+112.46\u0026thinsp;+\u0026thinsp;9.67*A\u0026thinsp;+\u0026thinsp;7.83*B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.75\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTS (MPa)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e=+17.07\u0026ndash;3.17*A-2.17*B\u0026thinsp;+\u0026thinsp;1.00*A*B-0.74*A2\u0026thinsp;+\u0026thinsp;2.26*B2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.82\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEAB (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e=+12.15\u0026thinsp;+\u0026thinsp;2.50*A\u0026thinsp;+\u0026thinsp;1.17*B-2.50*A*B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.91\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMoisture (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e=+31.38\u0026ndash;8.83*A-5.50*B\u0026thinsp;+\u0026thinsp;3.75*A*B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.95\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWVP (g \u0026micro;m/m\u003csup\u003e2\u003c/sup\u003e/d/ kPa)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e=+302.24\u0026ndash;79.67*A-20.00*B\u0026thinsp;+\u0026thinsp;16.75*A*B-34.34*A2\u0026thinsp;+\u0026thinsp;8.66*B2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Thickness, TS and EAB\u003c/h2\u003e \u003cp\u003eThe thickness of a biofilm or biomarker affects its mechanical properties. Also, the thickness of a film or membrane affects the permeability to water vapor and other gases. Mechanical resistance in films and markers used for smart food packaging is of great importance. Considering that biomarkers are usually inside the food and in direct contact with the food, it should have a suitable mechanical stability. In the current research, vitamin C and methylene blue used in the structure of composite film have the ability to control the oxidative and microbial spoilage of food products, especially oils, and therefore their thickness and mechanical resistance had to be checked.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows contour plot and perturbation curves of the effect of methylene blue amount and vitamin C percentage on the thickness, TS and EAB of cellulose films. Examining the thickness curves shows that both factors of methylene blue and vitamin C increase the thickness of the film. Considering that cellulosic films have porosity, the placement of methylene blue and vitamin C in these holes as well as the coating of these substances on the surface of cellulosic fibers increases the diameter of cellulosic fibers and ultimately leads to an increase in the thickness of the entire film. According to the results of TS and EAB, both factors, the amount of methylene blue and the percentage of vitamin C, have reduced the tensile strength and have increased the EAB and flexibility of the film. The effect of methylene blue on reducing the tensile strength and increasing the flexibility of the film has been greater than the effect of vitamin C. Due to the presence of N and S groups in methylene blue and O groups in the structure of vitamin C, the probability of electrostatic interactions and hydrogen bonds between these groups with the OH groups of cellulose is high, which makes these interactions of the cohesion of cellulose polymer chains weaker and reduces its tensile strength and probably increases flexibility for this reason. Shi et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) modified cellulose film with methylene blue and investigated its structure. The results of their research, from the point of view of the effect of methylene blue on the mechanical properties of cellulose film, confirm the results of the current research to some extent. Also, Atila et al. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) have reported the effect of vitamin C on the physical resistance and other characteristics of cellulosic fibers, and their results show relative agreement with the results of the current research.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Moisture content and WVP\u003c/h2\u003e \u003cp\u003eOne of the most important reasons for the spoilage of food products is the presence of moisture inside the package. Also, the penetration of unwanted gases and moisture into the food product can affect the shelf life of the food product. The characteristics of water resistance in markers that are used for smart packaging are also very important, because it can affect the sensitivity, efficiency and length of useful use of these markers. Based on this, in this work, the moisture content and water vapor permeability of the prepared composite films were investigated. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows contour plot and perturbation curves of the effect of methylene blue and vitamin C on moisture content and WVP of cellulose films. The results of checking the moisture content show that both methylene blue and vitamin C agents have significantly reduced the moisture content. By examining the chemical structure of cellulose, methylene blue and vitamin C, it can be estimated that N and S groups in methylene blue and O groups in vitamin C have electrostatic interactions with the OH groups of cellulose that this phenomenon decreases the hydrogen interactions between the OH groups of cellulose and the H\u003csub\u003e2\u003c/sub\u003eO molecule, which leads to a decrease in the amount of water molecules on the cellulose surface and the moisture content decreases.\u003c/p\u003e \u003cp\u003eAlso, both factors of methylene blue and vitamin C have reduced the WVP. As discussed earlier, cellulose has a porous structure that is susceptible to the passage of various gases and especially water vapor molecules, which with the composite of this film with methylene blue and vitamin C, these pores are filled to a large extent and the passage of water molecules is blocked and the WVP is reduced. Also, as discussed in the discussion of thickness, methylene blue and vitamin C increase the thickness of cellulose and thus increase the length of the passage of water molecules, which causes a decrease in permeability to water vapor. Tan et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) have investigated the water resistance and antioxidant properties of chitosan-ascorbate film modified with methylene blue. The results of their research in terms of the effect of methylene blue on the characteristics of water vapor permeability and other characteristics of water resistance are in relative agreement with the results of the current research.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Antioxidant and antibacterial property\u003c/h2\u003e \u003cp\u003eMethylene blue as a phenothiazine dye has the ability to stain biofilm. This pigment has antimicrobial effects. Methylene blue shows good antibacterial properties by absorbing light. The antibacterial effect of methylene blue is caused by DNA damage, but methylene blue is non-toxic in the human body (Calanna et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Also, methylene blue has oxazine or antioxidant properties. The antioxidant ability of methylene blue is due to the fact that it loses electrons in the presence of oxidizing agents. Vitamin C is effective in the survival, destruction and overall metabolism of various bacteria. Although bacteria usually have the ability to ferment vitamin C, this vitamin can expose bacteria to oxidative stress and inhibit bacterial growth (Mousavi et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eConsidering that methylene blue and vitamin C both have antioxidant and antimicrobial properties, in this study, the antioxidant and antimicrobial properties (against \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and \u003cem\u003eEscherichia coli\u003c/em\u003e) of cellulose films containing these two substances have been investigated. Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows the antioxidant and antibacterial properties of cellulose films containing methylene blue and vitamin C. As it is clear from the results, the pure cellulose film does not have antibacterial properties, but it has a very small amount of antioxidant properties, which is probably related to the physical removal of DPPH radicals by the cellulose film. Both methylene blue and vitamin C have increased antioxidant properties, and the film containing both methylene blue and vitamin C has the highest antioxidant properties. Of course, it is clear that the effect of vitamin C on antioxidant properties is much higher than that of methylene blue. However, vitamin C is a very strong and well-known antioxidant, and the results obtained were expected. Pure cellulose film does not show any antibacterial properties, but films containing vitamin C and methylene blue show good antibacterial properties against both types of bacteria (Gram positive and Gram negative). Cellulose film, which simultaneously contains methylene blue and vitamin C, shows the most antimicrobial properties, which indicates the synergy of the antibacterial effect of methylene blue and vitamin C. There are many studies that have reported the antibacterial properties of methylene blue and vitamin C, which confirm the results of the present study (Thesnaar et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Mumtaz et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntioxidant and antibacterial properties of cellulose film and its composites\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFilm\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eAntibacterial activity:\u003c/p\u003e \u003cp\u003eInhibition zone diameter (mm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAntioxidant activity (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"1\" nameend=\"c5\" namest=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e (G-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e (G+)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCel\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e4\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCel/MB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e42\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCel/VC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e64\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCel/MB/VC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e82\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e*Different letters in each column indicate the significance of the difference in means\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.4. SEM images and FTIR spectra\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows SEM images and FTIR spectra of cellulose films and its various composites. According to the SEM images, the pure cellulose film has a fibrous structure with dimensions of 20 to 100 nm, which has significant porosity on the surface of the film. By adding methylene blue on the film, the porosity of the film surface has been filled to a large extent and a uniform surface has been created. Vitamin C fills most of the surface of the fibers and in the cellulose film containing vitamin C, the surface porosity of the cellulose film is still observed. In the cellulose film containing methylene blue and vitamin C, the surface of the polymer is largely saturated with the additive and the penetration paths of various gases from the surface of the polymer are largely filled. Acharya et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) have investigated the structure of cellulosic film and modified cellulosic films by SEM technique, and their results are in good agreement with the results of the present research (Acharya et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eExamination of FTIR spectra confirms the electrostatic interactions between cellulose fibers, methylene blue and vitamin C. In the FTIR spectrum of pure cellulose film, the functional groups of this polymer have been confirmed by different peaks. In this spectrum, peak at 3330 cm\u003csup\u003e-1\u003c/sup\u003e indicates the stretching vibrations of O-H groups. Peak 2894 represents C-H vibrations in R-CH\u003csub\u003e2\u003c/sub\u003e-CH\u003csub\u003e3\u003c/sub\u003e structures. The 1415 peak confirms intra-ring C-C vibrations and the 1152 peak shows C-O stretching vibrations of the C-H bond. Peaks of 1023 and 875 are respectively related to C-O and C-H vibrations connected to different functional groups. By comparing the spectrum of pure cellulose film with its different composites, no significant difference is observed between the spectra because most of the hydrocarbon structure and elements in different composites are similar. But it can be seen that the peaks related to the same functional groups in different composites have appeared in different wave numbers (shifted to higher or lower wave numbers), which indicates the electrostatic interactions between the composite components. Also, in the spectrum of cellulose/methylene blue, a new peak has appeared (compared to the pure cellulose film spectrum) at wave number of 1640, which is related to C-N stretching vibrations, which was expected due to the structure of methylene blue. Also, in the spectrum of cellulose/vitamin C, two new peaks (compared to the pure cellulose film spectrum) have appeared at wave numbers of 1665 and 1751, which are related to C\u0026thinsp;=\u0026thinsp;C stretching vibrations specific to vitamin C. Hishikawa et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) investigated the FTIR spectrum of cellulose film, Ovchinnikov et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) investigated the chemical structure of methylene blue using FTIR spectrum and Voss et al. (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) investigated the FTIR spectrum of polysaccharide film containing vitamin C. The mentioned studies confirm the results of FTIR spectra of the present study.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.5. XRD spectra and TGA curves\u003c/h2\u003e \u003cp\u003eCrystalline structure and thermal resistance are characteristics of films that can affect other characteristics of films such as chemical and thermal stability. The type of interaction of additives with cellulose is largely related to the crystalline structure and porosity of cellulose. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e shows the XRD spectrum and the TGA curve of cellulose films and its various composites. Based on the XRD spectrum, the pure cellulose film has a semi-crystalline cellulose structure, which shows distinct peaks at 2θ of 15, 17 and 22 degrees, and these peaks indicate the crystalline structure of this film, which is in full agreement with the results reported in previous researches. By examining the cellulose film modified with methylene blue and vitamin C, it was found that methylene blue has no significant effect on the crystalline structure of cellulose, but vitamin C increased the crystalline properties of the cellulose film. In the cellulose film containing vitamin C, in addition to the peaks related to cellulose, several peaks at 2θ of 25, 27, 30 and 33 have appeared, all of which are related to the crystal structure of vitamin C. According to the total XRD spectra of different films, it can be concluded that methylene blue does not affect the crystalline nature of cellulose, but vitamin C improves the crystalline order of the cellulose film by coating the cellulose fibers. In the studies of Kumar et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and Ahmed et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), the XRD spectra of cellulose and vitamin C have been investigated, which confirm the results reported for the XRD spectra of the peaks recorded for cellulose and vitamin C in this study. Examining the TGA curves showed that the cellulose film undergoes weight decomposition in two stages. The first stage occurs at a temperature between 70 and 130\u0026deg;C, which is related to the evaporation of possible water molecules in the film structure. Weight loss at this stage is about 5%. The second stage of decomposition, which is related to the complete destruction of the polymer, takes place at a temperature between 280 and 400\u0026deg;C. At this stage, 90 to 95% of the polymer is destroyed and destroyed. By examining the TGA curves of composite films, it was found that by adding methylene blue and vitamin C to the film, the temperature of thermal decomposition increases, and in other words, the thermal resistance of the film is improved. The cellulose film containing both methylene blue and vitamin C has the highest decomposition temperature and the highest thermal resistance, which indicates the synergistic effect of these two substances. It can be said that the electrostatic interactions between the composite components, which were also mentioned in the analysis of the FTIR spectra, have led to the improvement of the thermal resistance of the cellulose film. Nurazzi et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) have investigated the thermal resistance and crystal structure of cellulose films and its composites, and their results are in good agreement with the results of the present research.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Absorption rate and release of methylene blue\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e shows the one-factor curve of methylene blue absorption rate on cellulose film and the three-dimensional curve of the effect of the initial amount of methylene blue and vitamin C concentration on the rate of release of methylene blue from cellulose films containing this substance. As it is known, the higher the amount of methylene blue used to prepare the composite film, the higher the amount of absorption on the film, which seems to be a natural result, because the higher the amount of methylene blue, the higher the amount of particles available to the cellulosic fibers. Also, the more methylene blue absorbed on the cellulose film, the higher its release rate will be. An important result that can be seen is that in the composite films where vitamin C is present along with methylene blue in the film, the amount of methylene blue release has decreased a lot. This result is due to the oxidation-reduction interaction of methylene blue and vitamin C, as well as vitamin C, which is added during the production stage after the stabilization of methylene blue on cellulose, acts like a coating and prevents its release. The point that should be noted is that the maximum release of methylene blue from films containing methylene blue and vitamin C was around 15%, which according to some sources reported that methylene blue was not recommended for oral consumption, it seems that this amount of release is problematic, but it should be noted that the methylene blue/vitamin C cellulose film is designed for the intelligent packaging of oily products to delay the oxidation of oils and also show its expiration time, but In this research, the rate of release in water is reported, and it is likely that the rate of release of methylene blue in oils will be much lower. In a similar research, Khakpour et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) have used starch film containing lycopene pigment as a nitrite detection kit, and the results of their research in terms of the application and performance of the sensor in identifying oxidants in food products show relative agreement with the results of the current research.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Application of film as H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e sensor (kit)\u003c/h2\u003e \u003cp\u003e4 films according to Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e1\u003c/span\u003e-C were used to check the performance of films to identify oxidizing agents (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e). To use the films as a kit, these films were cut in dimensions of 1\u0026times;4 cm. In these films, due to the fact that vitamin C is a reducing substance and due to the fact that methylene blue is colorless in its reduced form, the color of these films is white (the main color of cellulose as the base of the kit). Adding oxidizing agents such as hydrogen peroxide to the surface of the kit changes the color of the kit from white to blue. The amount and intensity of color changes of the sensor depend on the concentration of the oxidizing agent. Therefore, a mathematical relationship is established between the concentration of the oxidizing agent and the color changes, through which the prepared kits can be calibrated with respect to the oxidizing agents, and the prepared calibration curve can be used to obtain the amount and concentration of the oxidizing agent. In this study, hydrogen peroxide was used as a soft oxidizer to investigate the behavior of the prepared sensors. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e-B shows the color changes of the sensor in different concentrations of hydrogen peroxide. Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows the figure of merits of the 4 prepared kits compared to hydrogen peroxide.\u003c/p\u003e \u003cp\u003eAs it is clear from the results of this table, the cellulose film containing the highest amount of methylene blue and the highest amount of vitamin C shows the highest sensitivity to hydrogen peroxide oxidant. It is known that by increasing the amount of methylene blue on the surface of the film, the amount of this active substance that is available to the oxidant also increases, and causes even small amounts of the oxidant to cause significant color changes on the surface of the film, which causes The detection limit of the sensor to measure the oxidant should also be reduced. It should be mentioned that in the examination of sensors, the lower the detection limit of the sensor is, it indicates the high detection power of the sensor and the high sensitivity of the sensor. Jiang et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) have used silica/cellulose composite modified with catalytic enzymes as a sensor to measure hydrogen peroxide. Considering the use of enzyme and catalytic structure in the mentioned study, it should be noted that the present research has presented a simpler, cheaper and more accessible system compared to the research of Jiang et al. Also, Jiang et al.'s results show a good match with the results of the current research in terms of the application of the sensor and the functional system of the sensor.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFigure of merits of the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e Kits based on the b (color factor) compared to the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKit\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDL (mg/100ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLR (mg/100ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eS (100ml/mg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eK1 (Cel/MB50/VC2.5)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.12-22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1549\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.997\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eK2 (Cel/MB50/VC5)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.4\u0026ndash;25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1742\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.998\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eK3 (Cel/MB100/VC2.5)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u0026ndash;21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.2526\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.998\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eK4 (Cel/MB100/VC5)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.1\u0026ndash;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.2995\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.999\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eDL: Detection limit, LR: Liner range, S: Sensitivity, R\u003csup\u003e2\u003c/sup\u003e: Coefficient of determination\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn this research, an active and smart biodegradable film was designed. For this purpose, cellulose nanofiber film modified with methylene blue and vitamin C. In this research, hydrogen peroxide was used as a soft oxidant to investigate the sensing behavior of the prepared cellulose film. Using mathematical equations, linear relationships were established between the color changes of the sensor and the concentration of hydrogen peroxide, and it was used for qualitative and quantitative measurement of hydrogen peroxide. Examination of the thickness showed that both the amount of methylene blue and vitamin C increased the thickness of the film. Methylene blue and vitamin C decreased the tensile strength and increased EAB. Methylene blue and vitamin C agents significantly reduced the moisture content. Also, both factors reduced the amount of methylene blue and vitamin C decreased WVP. The pure cellulose film did not have antibacterial properties, but it had antioxidant properties to a very small extent. Both methylene blue and vitamin C increased the antioxidant properties. The pure cellulose film did not show any antimicrobial properties, but the films containing vitamin C and methylene blue showed good antimicrobial properties against both types of bacteria (Gram positive and Gram negative). The pure cellulose film had a fibrous structure with dimensions of 20 to 100 nm. By adding methylene blue on the film, the porosity of the film surface was filled to a large extent and a uniform surface was created. Examination of FTIR spectra confirmed the electrostatic interactions between cellulose fibers, methylene blue and vitamin C. Methylene blue had no significant effect on the crystalline structure of cellulose, but vitamin C increased the crystalline properties of cellulose film. The electrostatic interactions between the composite components, which were also proven in the FTIR spectrum analysis, have led to the improvement of the thermal resistance of the cellulose film. The highest rate of release of methylene blue from films containing methylene blue and vitamin C was about 15%. Cellulose film containing the highest amount of methylene blue and the highest amount of vitamin C showed the highest sensitivity to hydrogen peroxide oxidant. The prepared sensors were able to measure hydrogen peroxide with appropriate sensitivity and acceptable detection limits. Due to this feature, the prepared films will be used to detect the oxidation process in smart food packaging.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eEthical Approval\u003c/h2\u003e \u003cp\u003eEthical approval for the study was obtained from the relevant local ethics committees.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eConsent to Participate\u003c/h2\u003e \u003cp\u003eAll authors consent to participate in the research project\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent to Publish\u003c/strong\u003e \u003cp\u003eAll authors consent to publish the current manuscript.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eThere is not any Conflict of interest between authors.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eDeclaration\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eSajad Pirsa conceived of the presented idea, Narmela Asefi developed the theory and performed the computations. Sajad Pirsa verified the analytical methods. Sina Sadeghi discussed the results and contributed to the final manuscript. Sina Sadeghi out the experiment. Sajad Pirsa and Mehdi Gharekhani wrote the manuscript and revised it.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdolsattari, P., Peighambardoust, S. J., Pirsa, S., Fasihnia, S. H. \u0026amp; Peighambardoust, S. H. Investigating microbial properties of traditional Iranian white cheese packed in active LDPE films incorporating metallic and organoclay nanoparticles. \u003cem\u003eChem. Rev. Lett.\u003c/em\u003e \u003cb\u003e3\u003c/b\u003e (4), 168\u0026ndash;174 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbdolsattari, P., Rezazadeh-Bari, M. \u0026amp; Pirsa, S. 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Polysaccharide-based film loaded with vitamin C and propolis: A promising device to accelerate diabetic wound healing. \u003cem\u003eInt. J. Pharm.\u003c/em\u003e \u003cb\u003e552\u003c/b\u003e (1\u0026ndash;2), 340\u0026ndash;351 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYorghanlu, R. A., Hemmati, H., Pirsa, S. \u0026amp; Makhani, A. Production of biodegradable sodium caseinate film containing titanium oxide nanoparticles and grape seed essence and investigation of physicochemical properties. \u003cem\u003ePolym. Bull.\u003c/em\u003e \u003cb\u003e79\u003c/b\u003e (10), 8217\u0026ndash;8240 (2022).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Biodegradable film, Kit, oxidation/regeneration, Antioxidant, Antibacterial","lastPublishedDoi":"10.21203/rs.3.rs-5274857/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5274857/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn this study, cellulose nanofiber film was modified with methylene blue pigment and vitamin C (Cel/MB/VC). The physicochemical, structural, thermal resistance, and antibacterial characteristics of the prepared films were investigated using SEM, FTIR, XRD, TGA, etc. The rate of absorption and release of methylene blue from the prepared film was studied. Films containing methylene blue and vitamin C were used as a kit to detect hydrogen peroxide. The obtained results showed that methylene blue and vitamin C increased the thickness of the film and the elongation of the film. Both methylene blue and vitamin C agents reduced moisture content and water vapor permeability. Both methylene blue and vitamin C significantly increased the antioxidant and antimicrobial properties (against \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e) of the film. SEM images showed that the diameter of cellulose fibers is between 20 and 100 nm, which methylene blue and vitamin C completely cover their surface and the surface porosity of the film. FTIR spectra confirmed the electrostatic interactions between cellulose, methylene blue and vitamin C. According to the XRD results, cellulose has a crystalline structure, which vitamin C improved this crystalline property. According to TGA results, methylene blue and vitamin C caused more thermal stability of cellulose film. The release of methylene blue from the film was reported to be 15%. In the presence of hydrogen peroxide, the color of the films containing methylene blue and vitamin C changed from white to blue. Films containing methylene blue and vitamin C at the same time showed good performance as a hydrogen peroxide detection sensor (kit). The highest sensitivity of the sensor for measuring hydrogen peroxide was 0.299 (100 mg/ml) with a detection limit of 1.9 (100 mg/ml). The prepared film has the ability to be used in smart packaging of foods sensitive to oxidation such as oils.\u003c/p\u003e","manuscriptTitle":"Biodegradable film based on cellulose nanofiber/methylene blue/vitamin C: investigation of physicochemical properties and usability as a kit for identification of oxidants (hydrogen peroxide)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-20 11:07:48","doi":"10.21203/rs.3.rs-5274857/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-01-02T05:57:15+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-18T07:51:28+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-17T18:48:08+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-16T04:13:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"329824574498427431834719135799372697151","date":"2024-11-07T13:09:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"54500461965617327267269122788574902801","date":"2024-11-06T02:43:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"164542600207085880052625866301110621202","date":"2024-11-05T15:18:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"279814803086323541728053573470346775914","date":"2024-11-05T14:01:13+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-05T11:25:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-05T11:22:50+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-11-05T02:04:03+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-30T04:16:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-10-16T09:57:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"63528841-c999-4c38-b33e-a4d6e3811c07","owner":[],"postedDate":"November 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":39956113,"name":"Biological sciences/Biochemistry"},{"id":39956114,"name":"Health sciences/Biomarkers"},{"id":39956115,"name":"Physical sciences/Chemistry"}],"tags":[],"updatedAt":"2025-07-07T16:18:12+00:00","versionOfRecord":{"articleIdentity":"rs-5274857","link":"https://doi.org/10.1038/s41598-025-07566-6","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-07-02 15:58:45","publishedOnDateReadable":"July 2nd, 2025"},"versionCreatedAt":"2024-11-20 11:07:48","video":"","vorDoi":"10.1038/s41598-025-07566-6","vorDoiUrl":"https://doi.org/10.1038/s41598-025-07566-6","workflowStages":[]},"version":"v1","identity":"rs-5274857","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5274857","identity":"rs-5274857","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}