key: cord-0768501-chr0vgmv
authors: Tan, Ezgi; Kahyaoğlu, İbrahim Mizan; Karakuş, Selcan
title: A sensitive and smartphone colorimetric assay for the detection of hydrogen peroxide based on antibacterial and antifungal matcha extract silver nanoparticles enriched with polyphenol
date: 2021-08-14
journal: Polym Bull (Berl)
DOI: 10.1007/s00289-021-03857-w
sha: 59aa4f888fa5940ac1886cfadd22394a34848af2
doc_id: 768501
cord_uid: chr0vgmv

Current trends in scientific studies focus on the development of smartphone-based biosensors via green nanoparticle for clinical diagnosis, food, and environmental monitoring. In this study, we developed a novel portable smartphone-based biosensor via green dendrimer-coated matcha extract/silver nanoparticles (ME-Ag NPs) enriched with polyphenol for detecting hydrogen peroxide (H(2)O(2)). Also, we investigated the biological evaluation of the nanostructure as a safe preservative for use in biomedical applications. Ag NPs were prepared using a green sonochemical method and were characterized to determine surface and chemical properties by different techniques such as scanning electron microscopy—energy-dispersive X-ray, transmission electron microscope, Fourier transform infrared spectroscopy, atomic force microscopy, X-ray diffraction, and Brunauer–Emmett–Teller. Furthermore, antimicrobial and antifungal properties of ME-Ag NPs were investigated against pathogenic microorganisms such as Staphylococcus aureus, Pseudomonas aureginosa, Escherichia coli, Candida albicans, and Aspergillus brasiliensis. The experimental sensor methodology was based on the detection of H(2)O(2) by analysis of images of novel silver nanostructure-coated papers and processing of color histograms with a RGB (red–green–blue) analyzer software. Consequently, the smartphone-based biosensor exhibited high sensitivity with detection limits of 0.82 μM response time of 5 s. The smartphone-based biosensor via ME-Ag NPs provided a rapid and selective detection of H(2)O(2).

The monitoring and detection of H 2 O 2 have a significant role in biological organisms and a broad range of industrial applications [1] . For the determination of H 2 O 2 , different methods such as chromatography, electroanalysis, colorimetry, and fluorometry have been reported [2, 3] . Instead of bulky and expensive methods such as spectrophotometer, which are widely used to detection of target biochemical analytes, easy to use, portable, and inexpensive smartphone-based methods offer great advantages [4] In this case, it is significant to develop easyto-use and low-cost smartphone-based methods to detect H 2 O 2 with a high sensitivity and rapid detection at low concentration. In the last two decades, scientific studies focus on smartphone-based sensor technologies, which have great potential in studies for different purposes such as high-quality medical services, food control, diagnostic technologies, and environmental monitoring [5] [6] [7] . In particular, the use of smartphones as detectors has increased, which makes the detection method considerably practical and economical and facilitates its commercialization and spread in modern sensor technology. The smartphone is used as a detector, reflecting the colorimetric analysis using an image software-based histogram algorithm. The methodology of smartphone-based sensor is based on a highresolution camera and image software using mobile sensing approaches under a smartphone's flashlight or an external light source [3] . The digital image colorimetry method on a smartphone is known a fast and low-cost analysis method using a RGB analyzer software. A target analyte is (metal, drug, microorganism, and biomarkers) measured by color analysis of the digital image obtained using the built-in camera by the digital image colorimetry method [8] [9] [10] .

Recently, nanostructures have been widely used in the development of electrochemical biosensors to improve the biocompatibility, sensitivity, reproducibility, catalytic activity, and signal amplification [11] [12] [13] . With the remarkable advances and developments in nanotechnology, various nanomaterials such as nanolayers [14] , quantum dots [15] , NPs [16, 17] , carbon nanotubes [18] , nanofiber [19] , and nanocomposite [20] have been reported to be used in the development of electrochemical biosensors. Our aim was to develop the disposable colorimetric nanobiosensor paper and evaluate the color change with a smartphone. In this context, we designed a new colorimetric biosensor by integrating the nanostructure with a smartphone platform to apply the promising material in H 2 O 2 determination. In this study, we developed a novel smartphone-based biosensor via green dendrimer-coated matcha extract/silver nanoparticles (ME-Ag NPs) enriched with ployphenol for detecting H 2 O 2 based on digital RGB spectral responses. The smartphone-assisted method was based on nanoprobe-coated filter paper using RGB colorimetric approach. Additionally, the strategy of using smartphone-based biosensor to detect H 2 O 2 has not been reported and has unique advantages such as green, low-cost, portable, sensitivity, and easy to use.

In addition, biological evaluation of novel ME-Ag NPs was performed in this study. As known, current trends in scientific studies focus on the control the spreading and to prevent infections of microscopic organisms known as pathogens-such as coronavirus, cholera and plague or parasites [21] [22] [23] . One of the most promising developments of nanotechnolgy is the evaluation of preservative efficacy of nanoadditives, which have been gaining attention for preventing from pathogenic microbial contamination during the novel fabrication of cosmetics [24] [25] [26] [27] . Recently, metal/metal oxide-based nanoformulations are promising nanosized protectants against several pathogens in food, cosmetic, agriculture and drug with their excellent antimicrobial and antifungal properties [28] [29] [30] [31] [32] . Among these materials, green synthesized silver-based nanostructures are widely used to control bacterial growth in biomedical applications [33] . Ag-based nanostructures have a significant performance to penetrate cell walls of bacterial, changing the chemical structure of cell membrane of bacteria and even resulting in bacterial death due to their small size and biocompatibility, and excellent physicochemical properties [34, 35] . According to technological development and previous studies, several novel silver-based nanoformulations have been fabricated using different stabilizers such as chitosan [36] , alginate [37] , poly (methyl methacrylate) (PMMA) [38] , polyethylene glycol-polyvinyl alcohol (PEG-PVA) [39] , PVA/gum acacia [40] , polyvinylpyrrolidone (PVP) [41] , polylactic acid (PLA) [42] , clay [43] , and plant extract [44] . In particular, several studies have been proposed to explain the physicochemical and biological properties of plant extractbased Ag NPs in the literature. However, some disadvantages have been studied to control the miscibility, dispersion stability, and agglomeration of Ag-based formulation in liquids [45, 46] . With these disadvantages, desired outcomes have not been completely achieved. To overcome these problems, Ag NPs have been developed due to their large surface area, excellent dispersion due to then narrow size distribution, stable dispersion in aqueous solutions, and highly branched shaped structures to improve antimicrobial and antifungal properties. Another prominent aspect of this study was the use of low-cost, biodegradable matcha, which is grown in many countries of the world, as a green stabilizer in the synthesis of silver nanostructures.

For this purpose, we synthesized the hyperbranched unimolecular ME-Ag NPs (generation 3, G3) by the green and one-step sonochemical via a self-assembly process.

The surface characterization and functional groups of ME-Ag NPs were determined by different techniques such as SEM-EDX, TEM, FTIR AFM, XRD, and BET. Furthermore, we focused on the development of the hyperbranched dendrimer-coated silver nanoparticles (ME-Ag NPs) to establish a novel preservative nanoformulation for antifungal agents against microorganisms and mold-yeast in biomedical and cosmetic applications. We determined the preservative efficacy of ME-Ag NPs against pathogenic microorganisms such as S. aureus, P. aeruginosa, E. coli, C. albicans, and A. brasilliensis with lSO 11930:2012 guideline for the total aerobic mesophilic microorganism, and mold-yeast, respectively. Consequently, the biological results were examined comprehensively, the novel green nanostructure exhibited unique antifungal and antibacterial properties. Furthermore, the sensor results showed that the prepared smartphone-based biosensor is a promising system for development of the smartphone-based sensors for other contaminants.

Matcha powder (Camellia sinensis) purchased from Arifoğlu Company (Turkey). Sodium hydroxide, isopropyl alcohol, ethanol, ethyl acetate, and ammonia were purchased from Merck Company. Whatman® qualitative filter paper (diam. 25 mm) and silver nitrate (≥ 99.0%) were purchased from Sigma-Aldrich Company (Germany). Ascorbic acid (L-Ascorbic acid 99%), dopamine hydrochloride (98%), progesterone (4-Pregnene-3,20-dione, ≥ 99%), and glucose (D-(+)-Glucose monohydrate, 99.0%) were obtained from Sigma-Aldrich Company Ltd. (St Louis, USA). We used a sterile syringe filter with a 0.22-micron retention and polyvinylidene difluoride (PVDF) membrane. Ultrapure water was obtained from Merck Millipore Milli-Q® Integral 5. All chemicals and reagents were used without further purification.

First, ME powder was cleaned with distilled water to remove all impurities and then dried to constant weight at 50 °C in a vacuum heater for 2 h to remove residual moisture. Second, 10 g of the matcha powder was added to 500 ml of distilled water for 7 days at 25 °C in the dark medium at stable position. Finally, the solution was filtered using a sterile syringe filter to obtain ME. All samples were stored in sterile polypropylene containers until use at 25 °C.

The self-assembly sonochemical synthesis of ME-Ag NPs consisted of four steps. The first step, 0.42 g of AgNO 3 was dissolved in 250 ml of distilled water. Then, the solution was stirred for 10 min at 25 °C. 0.2 g of NaOH was dissolved in 125 ml of distilled water and stirred for 10 min at 25 °C. The second step, 50 ml of the ME and 100 ml of AgNO 3 solution, was sonicated for 5 min (amplitude frequency: 30%). The third step, 1 ml of NaOH solution was added slowly to the solution. We observed the color change of the sample. We assumed that ME had a role as a green reducing agent for reducing Ag + to Ag o by changing yellow to brown. Finally, ME-Ag NPs were filtered using a sterile syringe filter and stored in a sterile container until use at 25 °C in vacuum desiccator.

We used different advanced techniques such as scanning electron microscopy (SEM)-energy-dispersive X-ray (EDX), transmission electron microscope (TEM), Fourier transform infrared (FTIR) spectroscopy, atomic force microscopy (AFM), X-ray diffraction (XRD), and Brunauer-Emmett-Teller (BET) to characterize the nanostructure. The SEM-EDX (FEI QUANTA 450) (TedPella, double-coated, 8 mmW × 20 mL) was used to determine the surface morphology of the ME-Ag NPs with a 6-10 mm working distance, 0-130 Pa pressure, and voltage of 7-10 kV under low vacuum medium. Transmission electron microscope (TEM) (Hitachi HighTech HT7700) was used to visualize the ME-Ag NPs in a high vacuum mode at 100 kV. FTIR (Spectrum Two, Perkin Elmer,) was used to observe the functional groups of the samples (KBr powder) in the 4000-400 cm -1 frequency range with a resolution of 4 cm -1 and eight scans. The crystal structure and the mean size of crystallites of the ME-Ag NPs were determined by the XRD technique with Cu Kα radiation at 40 kV and 15 mA. The Brunauer-Emmett-Teller (BET) specific surface area of the sample was determined using a Micromeritics ASAP 2020. Absorbances of reference (NPs) and sample (NPs + analyte) were measured using a double-beam UV-Vis spectrophotometer (T + 80, PG Instrument) with a UVWin 5 Software.

The crystalline size of ME-Ag NPs was calculated using different XRD mathematical models such as the Scherrer, Williamson-Hall, and modified Scherrer equations, respectively (Eqs. 2.1-2.3) [47, 48] . The formulas of all models are given in Table 1 .

In this study, the storage stability of the synthesized ME-Ag NPs was evaluated under ultraviolet light. Five milliliters of ME-Ag NPs solution was irradiated at 365 nm for 2 h. We experimentally examined the effect of ultraviolet irradiation on the storage stability of the synthesized ME-Ag NPs by measuring the viscosity. Experimental rheological measurements were performed in triplicate.

In this study, the synthesized hyperbranched ME-Ag NPs enriched with polyphenol (generation 3, G3) were performed for antimicrobial and antifungal effect against different pathogenic microorganisms such as staphylococcus aureus (S. aureus, NCTC 10,788/Lot 0,350,520,029), Pseudomonas aureginosa (P. aeruginosa, ATCC 9027/Lot 3270513), Escherichia coli (E. coli, ATCC 8739/Lot 4835151), Candida albicans (C. albicans, NCPF 3179/Lot040920020), and Aspergillus brasilliensis (A. brasilliensis, NCPF 2275/Lot 020620065). 

The challenge test was performed using the standard NF EN lSO 11930:2012 guideline to evaluate the microbiological safety and level of biological activity of the preservative ME-Ag NPs (Table 2) . We examined by inoculation of the synthesized ME-Ag NPs with pathogenic microorganisms at a concentration range from 10 5 and 10 6 CFU g −1 corresponded to the colony-forming units per g of the synthesized ME-Ag NPs. Different pathogenic microorganisms were used such as Staphylococcus aureus (S. aureus, NCTC 10788/Lot 0350520029), Pseudomonas aureginosa (P. aeruginosa, ATCC 9027/Lot 3270513), Escherichia coli (E. coli, ATCC 8739/Lot 4835151), Candida albicans (C. albicans, NCPF 3179/Lot040920020), and Aspergillus brasilliensis (A. brasilliensis, NCPF 2275/Lot 020620065) and analyzed for challenging test of the synthesized ME-Ag NPs. All pathogenic microorganisms were issued from official collection of microbial strains of The European Union (EU) that ensures the reproducibility of the challenging tests. The inoculated ME-Ag NPs was held at temperature ranges from 20 to 25 °C in the dark medium for 28 days. The viability assessment was performed at suitable time intervals (T0 = 0 days (immediately after collection); T7 = 7 days; T14 = 14 days; and T28 = 28 storage days) with to the number of viable microorganisms. The preservative effectiveness test results were expressed in terms of the criteria A and B described in Table 3 .

In particular, the microbiological criteria were defined from decrease in logarithmic reduction scale 10 (log scale 10) of microorganism concentration from the measured inoculum using challenge test (according to the standard NF EN ISO 11930) for the evaluation of the antimicrobial protection of a cosmetic product. The result of challenge test gives two types of criteria such as a target (the "A" criterion) and an acceptable level (the "B" criterion). Finally, "no increase" was defined as not much more than 0.5 log10 units higher than the previous value measured.

The preparation steps of the biosensor arrays were as follows. (1) colorimetric solutions of ME-Ag NPs were dropped in different concentrations on a Whatman® qualitative filter paper using an Eppendorf® Research® plus pipette (0.1-2.5 μL), constructing a 2 cm diameter circular area of color-sensitive sensor array and (2) the sensor arrays were dried for 10 min and deposited in a sterile polyethylene bag before use. (3) H 2 O 2 solutions in the range from 0.05 to 20 μM were dropped on a ME-Ag NP-coated filter paper. (4) The images all samples were taken at room temperature with a smartphone (Casper F20) with a resolution of 1600 × 1200 pixels. Histograms of images of samples were obtained using a software (ImageJ 1.51q software) and analyzed the distribution of RGB values of samples. All experiments were performed in triplicate (n = 3).

The selectivity of the process was performed by detecting the effects of different analytes such as H 2 O 2 , isopropyl alcohol, ethanol, ethyl acetate, ammonia, ascorbic acid, dopamine hydrochloride, progesterone, and glucose in the response of the nanoprobe as in "Preparation of smartphone-based hydrogen peroxide biosensor" section.

In this study, we developed the novel portable smartphone-based biosensor via green ME-Ag NPs for detecting of H 2 O 2 . Furthermore, the detection mechanism of NPs biosensor was based on the characteristic color change of the nanocoated paper sensor, which was responded selective and rapid to target analyte using smartphone images of sample. Also, we detected the color change of samples and determined the H 2 O 2 concentration quantitatively using a spectroscopic measurement technique due to reaction with the target analyte.

All samples were imaged using a smartphone under sun light. We put the smartphone in a plexiglass glass-based box (8 cm × 8 cm and a 3-mm-thick clear acrylic sheets with a protective film) to keep it in a stable position. The colorimetric histograms of samples were determined to understand color differences in images using the RGB analysis with the software ImageJ. [49] . According to RGB analysis, values of color were assigned in the range from 0 to 255 for absolute black and absolute white, respectively.

where the subscripts i and 0 denote the RGB value (R: red, G: green, and B: blue) of the ME-Ag NPs with and without the analyte, respectively. The correlation between ΔE values and logH 2 O 2 concentration was obtained to determine the equilibrium time and colorimetric quantitative recognition. Furthermore, we calculated sensor

response data for ME-Ag NPs sensor papers to investigate their performance for sensor applications (Eq. 2.5) [50] .

where S: response in %, c: concentration, x c : sensor signal (RGB channels), x 0 : the value of x (c = 0) signal, and x ref : reference value. According to Eq. 2.6, we investigated the response of NPs paper sensor with the logarithm of the H 2 O 2 concentration [50] .

where m: constant and n: response at low concentration. Limit of detection (LOD) was known concentration of a component and calculated from calibration curve of the sample.

where S is the slope of the calibration curve and σ is the standard deviation of the response.

All experimental results were obtained in triplicate and are given as the mean for each experiment. The analysis of variance (ANOVA) procedure was conducted in SPSS (version 16, Chicago) with a significance level of P < 0.05.

In this study, ME was used as a stabilizing agent for hyperbranched unimolecular ME-Ag NPs (generation 3, G3). Surface techniques were used to control the size, dispersion, and stability of Ag NPs. Surface morphology and particle size of ME and ME-Ag NPs (G3-Ag) were investigated by SEM technique. SEM images of (a) pure matcha powder, (b) ME-Ag NPs with 40.000 × magnification, (c) ME-Ag NPs with 160.000 × magnification, (d) EDX spectra of ME-Ag NPs are given in Fig. 1 . The SEM result showed that pure matcha powder had an agglomerated and irregular shaped (Fig. 1a) [51, 52] . Figure 1b shows the SEM images of third generation ME-Ag NPs encapsulated with Ag nanoparticles which were uniformly anchored spheroidal dispersed within the polyphenolic dendrimer branching network. The size of Ag nanoparticles was found the ranges from 10 to 30 nm. According to SEM results, we showed the successful encapsulation of Ag nanoparticles within hyperbranched unimolecular polyphenolic dendrimer. The EDX spectra of ME-Ag NPs is shown in Fig. 1c . The characteristic absorption peaks of Ag o in the range of 1-2

(2.7) LOD = 3, 3 × ∕S Fig. 1 SEM images of a pure matcha powder, b ME-Ag NPs with 40.000 × magnification, c ME-Ag NPs with 160.000 × magnification, d EDX spectra of ME-Ag NPs and 3 keV were observed and the elemental EDX analysis also revealed C, O, and Ag [53] . The EDX results of ME-Ag NPs showed percentage relative composition of elements such as oxygen (O) 62.68%, carbon (C) 37.05%, and silver (Ag) 0.27%. According to BET results, we found that the surface area was 220.43 m 2 /g. The FTIR technique of (a) pure matcha powder and (b) ME-Ag NPs was used to investigate functional groups of structure in the region between 4000 and 400 cm −1 . FTIR analysis of pure matcha powder and ME-Ag NPs is shown in Fig. 2 . According to the FTIR results, the characteristic peaks of pure matcha powder were observed at 3278. 39 . These small shifts were due to the adsorption on the surface for C=O [55] . Consequently, we assumed that EGCG had a significant effect on reduction of Ag + in NPs. TEM technique was used to characterize the of the surface of ME-Ag NPs. Figure 2 shows the TEM images of (c) ME-Ag NPs x3k, (d) ME-Ag NPs x10k, and (e) ME-Ag NPs x30k with different magnifications. According to TEM results, we found that the NPs had a hyperbranched unimolecular structure due to the contribution of the Ag nanoparticles around the branches of the polyphenolic dendrimer. Briefly, we observed that 2D hyperbranched Ag nanoparticles in the lateral size range of about 10-20 nm were synthesized via a self-assembly sonication process at room temperature for 30 min using of green reducing agent, leading to the fabrication of dendrimer-branched Ag nanoparticles [56] . Also, Ag nanoparticles were well separated in the structure. The results were compatible with our SEM results. We assumed that the polyphenolic dendrimer homogenously stabilizes silver nanoparticles by branching the nanostructure.

X-ray diffraction (XRD) analysis of ME-Ag NPs was performed to calculate the crystalline size of Ag NPs in the structure. The XRD graph of the synthesized ME-Ag NPs is given in Fig. 3 . Figure 3 shows that characteristic peaks were formed around 2θ = 38.16°, 41.22°, 44.34°, 56.38°, 62.32°, 64.52°, and 64.84° corresponded to JCPDS file No.00-004-0783. We calculated the crystalline size of Ag NPs using different XRD mathematical models such as the Scherrer, modified Scherrer, and Williamson-Hall equations. The plot of (a) modified Scherrer and (b) Williamson-Hall of ME-Ag NPs is shown in Fig. 3 . The crystalline sizes of NPs were found 5.21 nm, 15.98 nm, and 15.74 nm by applying Scherrer, modified Scherrer, and Williamson-Hall formula, respectively (Fig. 3) . These results were well coincide with the SEM and TEM results. Also, these results were in good agreement with a related study [57] .

The synthesized ME-Ag NPs were characterized by AFM for three-dimensional morphology, topographical characterization, and agglomeration of Ag nanoparticles. The images were 5 µm × 5 µm. In Fig. 4 , (a) AFM topographical image, (b) threedimensional morphology, and (c) cross section of image of ME-Ag NPs were found to be in line with the SEM and TEM, showing the sizes of the synthesized ME-Ag NPs as 15 nm. The corresponding cross sections of the image was provided to show differences in height distributions between particles. According to AFM results, we observed that the ME-Ag NPs had a uniform size distribution and homogenous structure due to the polyphenolic network. Our results of AFM about surface topography of the synthesized ME-Ag NPs showed similarity to the results reported by many researchers concerning the surface characterization of Ag nanoparticles with uniform size distribution [58] [59] [60] [61] .

Various previous studies have proven the structure, content, and morphological properties of nanoparticles effect thermal conductivity, rheological behavior, viscosity, and stability [62] . Regarding the morphology-dependent rheological behavior and intrinsic viscosity measurement of nanofluids rich in Ag NPs, the lack of data in the literature is noteworthy. For this reason, we focused on the optimization of the viscosity of Ag NPs to have a long-term stability. The photographs of ME-Ag NPs after standing for 20 weeks (Fig. 5) showed that only a small amount of Ag NPs was precipitated at the bottom of the glass without obvious stratification, indicating their high storage stability. Furthermore, we found that Ag NPs had a high stable dispersion with small sizes in low concentrations and intrinsic viscosities of nanosystems. The storage stability of the hyperbranched unimolecular ME-Ag NPs was evaluated using rheological measurements. In Fig. 5 , the intrinsic viscosity/time plot of the ME-Ag NPs, and the schematic image of stability of SLE-Ag NPs at different times were given. The storage stability of the synthesized ME-Ag NPs was monitored by checking its intrinsic viscosity at 25 ± 0.1 °C. Also, the photograph of the ME-Ag NPs was taken over time (0-20 Week). Furthermore, the intrinsic viscosity difference of the ME-Ag NPs after 20 weeks of storage was found to be 34 ± 1.0 dl/g. These stability results exhibited an indication of its excellent storage stability with its photographs for the ME-Ag NPs. 

Recently, Coronavirus (COVID-19) is a significant issue and related to nanosized virus so we should improve the antimicrobial activity of novel materials and to prevent the spread of these pathogenic problems using nanotechnological solutions. With this viewpoint, we developed the novel ME-Ag NPs and investigated the biological activity of the NPs to evaluate the preservative efficacy of ME-Ag NPs against pathogenic microorganisms such as Staphylococcus aureus (S. aureus), Pseudomonas aureginosa (P. aeruginosa), Escherichia coli (E. coli), Candida albicans (C. albicans), and Aspergillus brasilliensis (A. brasilliensis), which cause infectious disease. These microorganisms are known major problems and cause infections to people or animals in direct or indirect routes [63, 64] . We used the total aerobic mesophilic microorganism (lSO 21149) and mold-yeast (lSO 16212) (cosmetics-microbiology-evaluation of the antimicrobial protection of a cosmetic product) guidelines for the antimicrobial and antifungal activity of the synthesized ME-Ag NPs. The preservative efficacy of ME-Ag NPs against pathogenic microorganisms of ME-Ag NPs were given in Fig. 6 . Concerning S. aureus, P. aeruginosa, E. coli, C. albicans, and A. brasilliensis, criterion A was satisfied for the synthesized ME-Ag NPs. To our knowledge, this report is the first study on preservative efficacy of the synthesized ME-Ag NPs against the S. aureus, P. aeruginosa, E. coli, C. albicans, and A. Brasilliensis for use in cosmetic preparations.

In previous studies, various reports were showed that plant extract-based nanoformulations had excellent biological activity against pathogenic microorganisms. In this study, our aim was to fabricate the biodegradable ME-Ag NPs without using any toxic reducing agents. The results showed that the synthesized Ag NPs had excellent antimicrobial and antifungal properties, and these results were appreciable from the comparison with the results of previous studies in the literature (Table 4) .

The smartphone-based ME-Ag NPs biosensor was developed for analysis of H 2 O 2 , isopropyl alcohol, ethanol, ethyl acetate, ammonia, ascorbic acid, dopamine hydrochloride, progesterone, and glucose. The analyte was deposited on the color region of ME-Ag NP-coated paper substrate and kept for 1 min to reach equilibrium. For color histogram analysis, the signal color intensity of ME-Ag NP-coated paper was calculated using the RGB color detector application in smartphone after addition of the target analyte. Furthermore, the limit of detection (LOD) was calculated using standard calibration curve with the response (S) (y)-axis and the Log H 2 O 2 concentrations on the horizontal (x)-axis. The selectivity detection of ME-Ag NPs toward various analytes, including H 2 O 2 , isopropyl alcohol, ethanol, ethyl acetate, ammonia, ascorbic acid, dopamine hydrochloride, progesterone, and glucose was conducted in the nanostructure-coated paper image with a smartphone and naked eye. Our experimental methodology was based on the fine adjustment of the color change with amount of the target analyte through pipetting attempts on the ME-Ag NP (10 μL NPs paste/cm 2 )-coated paper. All images (uncoated and coated paper) were captured from the top of the box using an autofocus mode in fixed viewing position between the detection smartphone camera and the biosensor (optimum distance: 8 cm) [71] . The analytical sensor performance of the smartphone-assisted biosensor for H 2 O 2 detection was experimentally done under the optimal condition such as room temperature, sun light, and Casper VIA F20 model smartphone. The smartphone has 48 + 5 + 2 + 2 MP quad rear camera, and the images of the samples were recorded in the continuum light source. With the camera supported by artificial intelligence, the samples were analyzed, and the best frames were captured [72] .

In Fig. 7 , the colorimetric analysis of smartphone camera-captured images of (a) ME-Ag NPs, and (b) ME-Ag NPs in the presence of H 2 O 2 , (c) ME-Ag NP-coated paper, (b) detection of H 2 O 2 using the ME-Ag NP-coated paper biosensor were presented. In the smartphone-supported sensor study, we observed that the mean value of RGB increased due to the presence of H 2 O 2 , but there was no significant change in the presence of other analytes (isopropyl alcohol, ethanol, ethyl acetate, ammonia, ascorbic acid, dopamine hydrochloride, progesterone, and glucose was conducted in the nanostructure-coated paper image with a smartphone and naked eye.), according to RGB analysis. In the RGB method, the digital images of the NPs sensor collected by a smartphone camera were divided into three channels: red (R), green (G), and blue (B) due to the color histogram results obtained using ImageJ software and the corresponding chromatic values [7] .

The small B value of NPs sensor showed the most remarkable response to H 2 O 2 . The B values were measured be 169, 96, 167, 147, 153, 145, 121, 98, 99, 97, and 100 for blank paper, ME-Ag NPs, ME-Ag NPs/H 2 O 2 , ME-Ag NPs /isopropyl alcohol, ME-Ag NPs/ethanol, ME-Ag NPs/ethyl acetate, ME-Ag NPs/ammonia, ME-Ag NPs/ascorbic acid, ME-Ag NPs/dopamine hydrochloride, ME-Ag NPs/progesterone, and ME-Ag NPs/glucose, respectively.

In this study, we investigated the logarithmic correlation between the H 2 O 2 concentration, and the response of the biosensor were determined from the calibration curve. The response (S) of sensor against logarithm of H 2 O 2 concentration in the range from 0.05 to 20 μM showed a linear relationship with a high correlation coefficient (R 2 ) of 0.9957. Based on experimental results of ME-Ag NP-coated paper biosensor, LOD was calculated using to the calibration curve and to be 0.82 μM with a signal-to-noise ratio (S/N = 3) in the linear dynamic range of 0.05-20 μM H 2 O 2 . We concluded that the developed smartphone-assisted method was successfully demonstrated for quantitative determination of H 2 O 2 .

The selectivity is known a significant characteristic of the sensor and the selectivity of sensor can be described using Euclidean distance (ΔE) values. The mathematical correlation between the calculated ΔE and analyte was shown in Fig. 7e values of sensor and a color change was observed with the naked eye. Furthermore, the color changes for all isopropyl alcohol, ethanol, ethyl acetate, and ammonia analytes were negligible. The concentration of NPs is a major factor in the sensitive detection of the target analyte with low concentrations which determine the economics sensor process. For this purpose, we analyzed the performance of the prepared ME-Ag NP-based sensor in different concentrations for detecting H 2 O 2 . Furthermore, we investigated the effect of concentrations of ME-Ag NPs in range of 0.5-10 μM on the colorimetric detection of hydrogen peroxide and colorimetric measurement results using colorimetric Euclidean distance (ΔE) values of smartphone camera-captured images. We found that the ME-Ag NP-based sensor exhibited an extremely high sensitivity to H 2 O 2 with a low concentration due to the strong interaction of H 2 O 2 in the sensitive nanoprobe-coated filter paper. Additionaly, the minimum Ag NPs concentration was determined as 2.3 μM for color change (Fig. 7i ). According to our experimental results, the estimated detection mechanism of H 2 O 2 is given in Eqs. 3.1-3.2.

A comparison of ME-Ag NP-based colorimetric H 2 O 2 sensor with other previous results were given in Table 5 . Our experimental results showed that the sensitive and low-cost ME-Ag NP-based sensor is suitable for colorimetric detection of H 2 O 2 .

Consequently, the ME-Ag NP-coated paper biosensor exhibited high selectivity for H 2 O 2 and acted as a H 2 O 2 -selective nanoprobe-coated filter paper using the smartphone-assisted method with the RGB colorimetric approach. It was clear that the fabricated H 2 O 2 sensing platform has a comparable LOD value than previous studies [2, 86, 87] . Using the smartphone-assisted color histogram analysis of image of ME-Ag NP-coated paper biosensor is a promising technique in sensor applications.

In this study, novel preservative nanostructure containing ME-Ag NPs enriched with polyphenol were prepared using a green and one-step sonochemical method via a self-assembly process. The antimicrobial and antifungal properties of the novel nanostructure was investigated against pathogenic microorganisms such as S. aureus, P. aeruginosa, E. coli, C. albicans, and A. brasilliensis. Characterization results showed that the synthesized ME-Ag NPs had a hyperbranched unimolecular, narrow size distribution (10 nm to 30 nm), and large surface area (220.43 m 2 /g). Furthermore, naked eye and colorimetric measurements confirmed the significant color change for the sensing H 2 O 2 using the smartphone-based ME-Ag NPs biosensors with the LOD of 0.82 μM, thereby it was found to be a low-cost, portable, and selective sensor for the determination of H 2 O 2 using the color histogram analysis.

(3.1) 2ME-AgNPs + H 2 O 2 ↔ 2ME-AgNPs … OH ad (3.2) ME-AgNPs … OH ad + e − + H + ↔ ME-AgNPs + H 2 O Fig. 7 The colorimetric analysis of smartphone camera-captured images of a ME-Ag NPs, and b ME-Ag NPs in the presence of H 2 O 2 , c ME-Ag NP-coated paper, d detection of H 2 O 2 using the ME-Ag NPcoated paper biosensor, e selectivity analysis of biosensor, f ΔE-log C H2O2 graph, g standard calibration of curve h Uv-Vis measurements of ME-Ag NPs/H 2 O 2 , i the effect of concentration of ME-Ag NPs We concluded that the challenge test proved that it had an excellent antibacterial and antifungal activity with criteria A data in cosmetic applications. 

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Acknowledgements This work was supported by Scientific Research Projects Coordination Unit of Istanbul University-Cerrahpasa. Project number 32438.

The authors declare no conflict of interests.